Lettuce cultivar Valley Fresh

ABSTRACT

A novel romaine lettuce cultivar, designated Valley Fresh, is disclosed. The invention relates to the seeds of lettuce cultivar Valley Fresh, to the plants of lettuce cultivar Valley Fresh and to methods for producing a lettuce plant by crossing the cultivar Valley Fresh with itself or another lettuce cultivar. The invention further relates to methods for producing a lettuce plant containing in its genetic material one or more transgenes and to the transgenic plants produced by that method and to methods for producing other lettuce cultivars derived from the cultivar Valley Fresh.

BACKGROUND OF THE INVENTION

The present invention relates to a new and distinctive Romaine lettuce (Lactuca sativa) cultivar, designated Valley Fresh. All publications cited in this application are herein incorporated by reference.

There are numerous steps in the development of any novel, desirable plant germplasm. Plant breeding begins with the analysis and definition of problems and weaknesses of the current germplasm, the establishment of program goals, and the definition of specific breeding objectives. The next step is selection of germplasm that possess the traits to meet the program goals. The goal is to combine in a single variety or hybrid an improved combination of desirable traits from the parental germplasm. These important traits may include increased head size and weight, higher seed yield, improved color, resistance to diseases and insects, tolerance to drought and heat, and better agronomic quality.

Practically speaking, all cultivated forms of lettuce belong to the highly polymorphic species Lactuca sativa that is grown for its edible head and leaves. As a crop, lettuces are grown commercially wherever environmental conditions permit the production of an economically viable yield. Lettuce is the world=s most popular salad. In the United States, the principal growing regions are California and Arizona which produce approximately 287,000 acres out of a total annual acreage of more than 300,000 acres (USDA, 2001). Fresh lettuces are available in the United States year-round although the greatest supply is from May through October. For planting purposes, the lettuce season is typically divided into three categories, early, mid and late, with the coastal areas planting from January to August, and the desert regions from August to December. Fresh lettuces are consumed nearly exclusively as fresh, raw product, occasionally as a cooked vegetable.

Lactuca sativa is in the Cichoreae tribe of the Asteraceae (Compositae family). Lettuce is related to chicory, sunflower, aster, dandelion, artichoke and chrysanthemum. Sativa is one of about 300 species in the genus Lactuca. There are seven different morphological types of lettuces. The Crisphead group includes the iceberg and batavian types. Iceberg lettuce has a large, firm head with a crisp texture and a white or creamy yellow interior. Batavian lettuce predates the iceberg type and has a smaller and less firm head. The Butterhead group has a small, soft head with an almost oily texture. Romaine lettuce, also known as cos lettuce, has elongated upright leaves forming a loose, loaf shaped head. The outer leaves are usually dark green. The Leaf lettuces come in many varieties, none of which form a head. The next three types are seldom seen in the United States: Latin lettuce looks like a cross between romaine and butterhead; stem lettuce has long, narrow leaves and thick, edible stems, and Oilseed lettuce is a type grown for its large seeds that are pressed to obtain oil.

Lactuca sativa is a simple diploid species with nine pairs of chromosomes. Lettuce is an obligate self-pollinating species. This means that the pollen is shed before stigma emergence, assuring 100% self-fertilization. Since each lettuce flower is an aggregate of about 10-20 individual florets (typical of the Compositae family), manual removal of the anther tubes containing the pollen is tedious. As such, a modified method of misting to wash the pollen off prior to fertilization is needed to assure crossing or hybridization. About 60-90 min past sunrise, flowers to be used for crossings are selected. The basis for selection are open flowers, with the stigma emerged and the pollen visibly attached to the single stigma (about 10-20 stigma). Using 3-4 pumps of water from a regular spray bottle, the pollen grains are washed off with enough pressure to dislodge the pollen grains, but not enough to damage the style. Excess water is dried off with clean paper towels. About 30 min later the styles should spring back up and the two lobes of the stigma are visibly open in a “V” shape. Pollen from another variety or donor parent is then introduced by gently rubbing the stigma and style of the donor parent to the maternal parent. Tags with the pertinent information on date and pedigree are then secured to the flowers

Choice of breeding or selection methods depends on the mode of plant reproduction, the heritability of the trait(s) being improved, and the type of cultivar used commercially (e.g., F₁ hybrid cultivar, pureline cultivar, etc.). For highly heritable traits, a choice of superior individual plants evaluated at a single location will be effective, whereas for traits with low heritability, selection should be based on mean values obtained from replicated evaluations of families of related plants. Popular selection methods commonly include pedigree selection, modified pedigree selection, mass selection, and recurrent selection.

The complexity of inheritance influences choice of breeding method. Backcross breeding is used to transfer one or a few favorable genes for a highly heritable trait into a desirable cultivar. This approach has been used extensively for breeding disease-resistant cultivars. Various recurrent selection techniques are used to improve quantitatively inherited traits controlled by numerous genes. The use of recurrent selection in self-pollinating crops depends on the ease of pollination, the frequency of successful hybrids from each pollination, and the number of hybrid offspring from each successful cross

Each breeding program should include a periodic, objective evaluation of the efficiency of the breeding procedure. Evaluation criteria vary depending on the goal and objectives, but should include gain from selection per year based on comparisons to an appropriate standard, overall value of the advanced breeding lines, and number of successful cultivars produced per unit of input (e.g., per year, per dollar expended, etc.). Promising advanced breeding lines are thoroughly tested and compared to appropriate standards in environments representative of the commercial target area(s) for three years at least. The best lines are candidates for new commercial cultivars; those still deficient in a few traits are used as parents to produce new populations for further selection. These processes, which lead to the final step of marketing and distribution, usually take from eight to 12 years from the time the first cross is made. Therefore, development of new cultivars is a time-consuming process that requires precise forward planning, efficient use of resources, and a minimum of changes in direction.

A most difficult task is the identification of individuals that are genetically superior, because for most traits the true genotypic value is masked by other confounding plant traits or environmental factors. One method of identifying a superior plant is to observe its performance relative to other experimental plants and to a widely grown standard cultivar. If a single observation is inconclusive, replicated observations provide a better estimate of its genetic worth.

The goal of lettuce plant breeding is to develop new, unique and superior lettuce cultivars. The breeder initially selects and crosses two or more parental cultivars, followed by repeated selfing and selection, producing many new genetic combinations. The breeder can theoretically generate billions of different genetic combinations via crossing, selfing and mutations. The breeder has no direct control at the cellular level. Therefore, two breeders will never develop the same cultivar, or even very similar cultivars, having the same lettuce traits.

Each year, the plant breeder selects the germplasm to advance to the next generation. This germplasm is grown under unique and different geographical, climatic and soil conditions and further selections are then made, during and at the end of the growing season. The cultivars that are developed are unpredictable because the breeder's selection occurs in unique environments, with no control at the DNA level (using conventional breeding procedures), and with millions of different possible genetic combinations being generated. A breeder of ordinary skill in the art cannot predict the final resulting cultivars he develops, except possibly in a very gross and general fashion. The same breeder cannot produce the same cultivar twice by using the exact same original parents and the same selection techniques. This unpredictability results in the expenditure of large research monies to develop superior lettuce cultivars.

The development of commercial lettuce cultivars requires the development of lettuce varieties, the crossing of these varieties, and the evaluation of the crosses. Pedigree breeding and recurrent selection breeding methods are used to develop cultivars from breeding populations. Breeding programs combine desirable traits from two or more varieties or various broad-based sources into breeding pools from which cultivars are developed by selfing and selection of desired phenotypes. The new cultivars are crossed with other varieties and the hybrids from these crosses are evaluated to determine which have commercial potential.

Pedigree breeding is used commonly for the improvement of self-pollinating crops or inbred cultivars of cross-pollinating crops. Two parents which possess favorable, complementary traits are crossed to produce an F₁. An F₂ population is produced by selfing one or several F₁s or by intercrossing two F₁s (sib mating). Selection of the best individuals is usually begun in the F₂ population; then, beginning in the F₃, the best individuals in the best families are selected. Replicated testing of families, or hybrid combinations involving individuals of these families, often follows in the F₄ generation to improve the effectiveness of selection for traits with low heritability. At an advanced stage of inbreeding (i.e., F₆ and F₇), the best cultivars or mixtures of phenotypically similar cultivars are tested for potential release as new cultivars.

Mass and recurrent selections can be used to improve populations of either self- or cross-pollinating crops. A genetically variable population of heterozygous individuals is either identified or created by intercrossing several different parents. The best plants are selected based on individual superiority, outstanding progeny, or excellent combining ability. The selected plants are intercrossed to produce a new population in which further cycles of selection are continued.

Backcross breeding has been used to transfer genes for a simply inherited, highly heritable trait into a desirable homozygous cultivar or line that is the recurrent parent. The source of the trait to be transferred is called the donor parent. After the initial cross, individuals possessing the phenotype of the donor parent are selected and repeatedly crossed (backcrossed) to the recurrent parent. The resulting plant is expected to have the attributes of the recurrent parent (e.g., cultivar) and the desirable trait transferred from the donor parent.

The single-seed descent procedure in the strict sense refers to planting a segregating population, harvesting a sample of one seed per plant, and using the one-seed sample to plant the next generation. When the population has been advanced from the F₂ to the desired level of inbreeding, the plants from which cultivars are derived will each trace to different F₂ individuals. The number of plants in a population declines each generation due to failure of some seeds to germinate or some plants to produce at least one seed. As a result, not all of the F₂ plants originally sampled in the population will be represented by a progeny when generation advance is completed.

Descriptions of other breeding methods that are commonly used for different traits and crops can be found in one of several reference books (e.g., “Principles of Plant Breeding” John Wiley and Son, pp. 115-161, 1960; Allard, 1960; Simmonds, 1979; Sneep et al., 1979; Fehr, 1987).

Proper testing should detect any major faults and establish the level of superiority or improvement over current cultivars. In addition to showing superior performance, there must be a demand for a new cultivar that is compatible with industry standards or which creates a new market. The introduction of a new cultivar will incur additional costs to the seed producer, the grower, processor and consumer for special advertising and marketing, altered seed and commercial production practices, and new product utilization. The testing preceding release of a new cultivar should take into consideration research and development costs as well as technical superiority of the final cultivar. For seed-propagated cultivars, it must be feasible to produce seed easily and economically.

Lettuce in general and romaine lettuce in particular is an important and valuable vegetable crop. Thus, a continuing goal of lettuce plant breeders is to develop stable, high yielding lettuce cultivars that are agronomically sound. To accomplish this goal, the lettuce breeder must select and develop lettuce plants with traits that result in superior cultivars.

The foregoing examples of the related art and limitations related therewith are intended to be illustrative and not exclusive. Other limitations of the related art will become apparent to those of skill in the art upon a reading of the specification.

SUMMARY OF THE INVENTION

The following embodiments and aspects thereof are described and illustrated in conjunction with systems, tools and methods which are meant to be exemplary and illustrative, not limiting in scope. In various embodiments, one or more of the above-described problems have been reduced or eliminated, while other embodiments are directed to other improvements.

According to the invention, there is provided a novel romaine lettuce cultivar designated Valley Fresh. This invention thus relates to the seeds of lettuce cultivar Valley Fresh, to the plants of lettuce cultivar Valley Fresh and to methods for producing a lettuce plant produced by crossing the lettuce Valley Fresh with itself or another lettuce cultivar, and to methods for producing a lettuce plant containing in its genetic material one or more transgenes and to the transgenic lettuce plants produced by that method. This invention also relates to methods for producing other lettuce cultivars derived from lettuce cultivar Valley Fresh and to the lettuce cultivar derived by the use of those methods. This invention further relates to hybrid lettuce seeds and plants produced by crossing the cultivar Valley Fresh with another lettuce cultivar.

In another aspect, the present invention provides regenerable cells for use in tissue culture of lettuce cultivar Valley Fresh. The tissue culture will preferably be capable of regenerating plants having the physiological and morphological characteristics of the foregoing lettuce plant, and of regenerating plants having substantially the same genotype as the foregoing lettuce plant. Preferably, the regenerable cells in such tissue cultures will be embryos, protoplasts, seeds, callus, pollen, leaves, anthers, pistils, roots, root tips and meristematic cells. Still further, the present invention provides lettuce plants regenerated from the tissue cultures of the invention.

Another aspect of the invention is to provide methods for producing other lettuce plants derived from lettuce cultivar Valley Fresh. Lettuce cultivars derived by the use of those methods are also part of the invention.

The invention also relates to methods for producing a lettuce plant containing in its genetic material one or more transgenes and to the transgenic lettuce plant produced by that method.

In another aspect, the present invention provides for single gene converted plants of Valley Fresh. The single transferred gene may preferably be a dominant or recessive allele. Preferably, the single transferred gene will confer such traits as male sterility, herbicide resistance, insect resistance, resistance for bacterial, fungal, or viral disease, male fertility, enhanced nutritional quality and industrial usage. The single gene may be a naturally occurring lettuce gene or a transgene introduced through genetic engineering techniques.

The invention further provides methods for developing lettuce plants in a lettuce plant breeding program using plant breeding techniques including recurrent selection, backcrossing, pedigree breeding, restriction fragment length polymorphism enhanced selection, genetic marker enhanced selection and transformation. Seeds, lettuce plants, and parts thereof produced by such breeding methods are also part of the invention.

In addition to the exemplary aspects and embodiments described above, further aspects and embodiments will become apparent by study of the following descriptions.

DEFINITIONS

In the description and tables which follow, a number of terms are used. In order to provide a clear and consistent understanding of the specification and claims, including the scope to be given such terms, the following definitions are provided:

Allele. The allele is any of one or more alternative form of a gene, all of which alleles relate to one trait or characteristic. In a diploid cell or organism, the two alleles of a given gene occupy corresponding loci on a pair of homologous chromosomes.

Backcrossing. Backcrossing is a process in which a breeder repeatedly crosses hybrid progeny back to one of the parents, for example, a first generation hybrid F₁ with one of the parental genotype of the F₁ hybrid.

Essentially all the physiological and morphological characteristics. A plant having essentially all the physiological and morphological characteristics means a plant having essentially all of the physiological and morphological characteristics of the recurrent parent, except for the characteristics derived from the converted gene.

Regeneration. Regeneration refers to the development of a plant from tissue culture.

Single gene converted. Single gene converted or conversion plant refers to plants which are developed by a plant breeding technique called backcrossing wherein essentially all of the desired morphological and physiological characteristics of a cultivar are recovered in addition to the single gene transferred into the cultivar via the backcrossing technique or via genetic engineering.

Maturity Date. Maturity refers to the stage when the plants are of full size or optimum weight, in marketable form or shape to be of commercial or economic value. In romaine types they range from 50-75 days from time of seeding, depending upon the season of the year.

RHS. RHS refers to the Royal Horticultural Society of England which publishes an official botanical color chart quantitatively identifying colors according to a defined numbering system, The chart may be purchased from Royal Horticulture Society Enterprise Ltd RHS Garden; Wisley, Woking; Surrey GU236QB, UK.

Yield (Tons/Acre). The yield in tons/acre is the actual yield of the lettuce at harvest.

DETAILED DESCRIPTION OF THE INVENTION

Lettuce cultivar Valley Fresh has superior characteristics and was developed in 2000 from the cross of the female parent Valmaine (unpatented) and White Heart Cos (unpatented) with the male parent Green Forest (U.S. Pat. No. 6,649,815). Six true F₁ plants were identified in the fall of 2000 in Salinas, Calif. The plants were allowed to self and the seeds were collected individually. A population of 400 F₂ plants were established in the spring of 2001 in Salinas, Calif. and 12 individual plants were selected. In the spring of 2002 nine F₃ families were planted and 14 individuals were selected. In the spring of 2003, 14 F₄ families were established and 18 individual plants were selected from 6 of the 14 F₄ families. In the spring of 2004, 12 F₅ lines were established and 22 individual plants were selected from six of the 12 F₅ lines. In the spring of 2005 eight F₆ lines were planted, line 05BS-0982 showed high uniformity and very desirable horticultural characteristics. In the summer of 2005, line 05BS-0982 was planted in a seed increase nursery in the San Joaquin Valley, Calif. and the seeds were harvested. Line 05BS-0982 became lettuce cultivar Valley Fresh.

Valley Fresh is a romaine lettuce with a medium-dark green leaf color, thick and moderate blistered leaf texture, large, moderately firm and slightly V-shaped head; it is widely adoptable in a variety of environments. Valley Fresh is highly resistant to corky root rot, tipburn, Schlertinia rot and brown rib. Valley Fresh has shown a very good adaptability in the California and Arizona regions of the United States.

Some of the criteria used for selection in various generations include: color, disease resistance, head weight, number of leaves, appearance and length, yield, emergence, maturity, plant architecture, seed yield and quality.

The cultivar has shown uniformity and stability for the traits, within the limits of environmental influence for the traits. It has been self-pollinated a sufficient number of generations with careful attention to uniformity of plant type. The cultivar has been increased with continued observation for uniformity. No variant traits have been observed or are expected in Valley Fresh.

Lettuce cultivar Valley Fresh has the following morphologic and other characteristics (based primarily on data collected at Salinas, Calif.).

TABLE 1 VARIETY DESCRIPTION INFORMATION Plant Type: Romaine Seed: Color: Black Light dormancy: Light not required Heat dormancy: Susceptible Cotyledon to Fourth Leaf Stage: Shape of cotyledons: Broad Undulation: Flat Anthocyanin distribution: Absent Rolling: Absent Cupping: Uncupped Reflexing: None Mature Leaves: Margin—Incision depth: Absent/Shallow Margin—Indentation: Absent/Slight Margin—Undulation of the apical margin: Absent/slight Green color: Medium-dark green Anthocyanin Distribution: Absent Size: Large Glossiness: Dull Blistering: Moderate Trichomes: Absent Leaf thickness: Thick Plant at Market Stage: Head shape: Slight V-shaped Head size class: Large Head weight:  775 g Head firmness: Moderate Core: Diameter at base of head: 3.83 cm Core height from base of head to apex: 7.16 cm Maturity: Summer:   57 days Winter:   98 days Adaptation: Primary Regions of Adaptation (tested and proven adapted) Southwest (California, Arizona desert): Adapted West Coast: Adapted Southeast: N/A Northeast: N/A Spring area: San Joaquin, CA; Imperial, CA; Yuma, AZ Summer area: Salinas, CA; Santa Maria, CA; San Benito, CA Fall area: Salinas, CA; Santa Maria, CA; Oxnard, CA Winter area: Yuma, AZ; Imperial, CA; Coachella, CA Greenhouse: N/A Soil Type: Both Mineral and Organic Disease and Stress Reactions: Virus: Big Vein: Intermediate Lettuce Mosaic: Susceptible Cucumber Mosaic: Not tested Broad Bean Wilt: Not tested Turnip Mosaic: Not tested Best Western Yellows: Not tested Lettuce Infectious Yellows: Not tested Fungal/Bacterial: Corky Root Rot (Pythium Root Rot): Highly resistant Downy Mildew: Susceptible Powdery Mildew: Susceptible Sclerotinia Rot: Highly resistant Bacterial Soft Rot (Pseudomonas sp. & others): Not tested Botrytis (Gray Mold): Susceptible Insects: Cabbage Loopers: Susceptible Root Aphids: Susceptible Green Peach Aphid: Susceptible Physiological/Stress: Tipburn: Highly resistant Heat: Intermediate Drought: Not tested Cold: Resistant Salt: Not tested Brown Rib: Highly resistant Post Harvest: Pink Rib: Resistant Russet Spotting: Not tested Rusty Brown Discoloration: Resistant Internal Rib Necrosis (Blackheart, Gray Rib, Resistant Gray Streak): Brown Stain: Not tested

FURTHER EMBODIMENTS OF THE INVENTION

This invention also is directed to methods for producing a lettuce cultivar plant by crossing a first parent lettuce plant with a second parent lettuce plant wherein either the first or second parent lettuce plant is a lettuce plant of the cultivar Valley Fresh. Further, both first and second parent lettuce plants can come from the cultivar Valley Fresh. Still further, this invention also is directed to methods for producing a cultivar Valley Fresh-derived lettuce plant by crossing cultivar Valley Fresh with a second lettuce plant and growing the progeny seed, and repeating the crossing and growing steps with the cultivar Valley Fresh-derived plant from 0 to 7 times. Thus, any such methods using the cultivar Valley Fresh are part of this invention: selfing, backcrosses, hybrid production, crosses to populations, and the like. All plants produced using cultivar Valley Fresh as a parent are within the scope of this invention, including plants derived from cultivar Valley Fresh. Advantageously, the cultivar is used in crosses with other, different, cultivars to produce first generation (F₁) lettuce seeds and plants with superior characteristics.

As used herein, the term plant includes plant cells, plant protoplasts, plant cell tissue cultures from which lettuce plants can be regenerated, plant calli, plant clumps and plant cells that are intact in plants or parts of plants, such as embryos, pollen, ovules, flowers, seeds, roots, anthers, pistils and the like.

As is well known in the art, tissue culture of lettuce can be used for the in vitro regeneration of a lettuce plant. Tissue culture of various tissues of lettuces and regeneration of plants therefrom is well known and widely published. For example, reference may be had to Teng et al., HortScience. 1992, 27: 9, 1030-1032, Teng et al., HortScience. 1993, 28: 6, 669-1671, Zhang et al., Journal of Genetics and Breeding. 1992, 46: 3, 287-290, Webb et al., Plant Cell Tissue and Organ Culture. 1994, 38: 1, 77-79, Curtis et al., Journal of Experimental Botany. 1994, 45: 279, 1441-1449, Nagata et al., Journal for the American Society for Horticultural Science. 2000, 125: 6, 669-672. It is clear from the literature that the state of the art is such that these methods of obtaining plants are “conventional” in the sense that they are routinely used and have a very high rate of success. Thus, another aspect of this invention is to provide cells which upon growth and differentiation produce lettuce plants having the physiological and morphological characteristics of variety Valley Fresh.

With the advent of molecular biological techniques that have allowed the isolation and characterization of genes that encode specific protein products, scientists in the field of plant biology developed a strong interest in engineering the genome of plants to contain and express foreign genes, or additional, or modified versions of native, or endogenous, genes (perhaps driven by different promoters) in order to alter the traits of a plant in a specific manner. Such foreign additional and/or modified genes are referred to herein collectively as “transgenes”. Over the last fifteen to twenty years several methods for producing transgenic plants have been developed, and the present invention, in particular embodiments, also relates to transformed versions of the claimed line.

Plant transformation involves the construction of an expression vector that will function in plant cells. Such a vector comprises DNA comprising a gene under control of or operatively linked to a regulatory element (for example, a promoter). The expression vector may contain one or more such operably linked gene/regulatory element combinations. The vector(s) may be in the form of a plasmid, and can be used alone or in combination with other plasmids, to provide transformed lettuce plants, using transformation methods as described below to incorporate transgenes into the genetic material of the lettuce plant(s).

Expression Vectors for Lettuce Transformation: Marker Genes

Expression vectors include at least one genetic marker, operably linked to a regulatory element (a promoter, for example) that allows transformed cells containing the marker to be either recovered by negative selection, i.e., inhibiting growth of cells that do not contain the selectable marker gene, or by positive selection, i.e., screening for the product encoded by the genetic marker. Many commonly used selectable marker genes for plant transformation are well known in the transformation arts, and include, for example, genes that code for enzymes that metabolically detoxify a selective chemical agent which may be an antibiotic or a herbicide, or genes that encode an altered target which is insensitive to the inhibitor. A few positive selection methods are also known in the art.

One commonly used selectable marker gene for plant transformation is the neomycin phosphotransferase II (nptII) gene, isolated from transposon Tn5, which when placed under the control of plant regulatory signals which confers resistance to kanamycin (Fraley et al., Proc. Natl. Acad. Sci. U.S.A., 80:4803 (1983)). Another commonly used selectable marker gene is the hygromycin phosphotransferase gene which confers resistance to the antibiotic hygromycin (Vanden Elzen et al., Plant Mol. Biol., 5:299 (1985)).

Additional selectable marker genes of bacterial origin that confer resistance to antibiotics include gentamycin acetyl transferase, streptomycin phosphotransferase, aminoglycoside-3′-adenyl transferase, the bleomycin resistance determinant (Hayford et al., Plant Physiol. 86:1216 (1988), Jones et al., Mol. Gen. Genet., 210:86 (1987), Svab et al., Plant Mol. Biol. 14:197 (1990), Hille et al., Plant Mol. Biol. 7:171 (1986)). Other selectable marker genes confer resistance to herbicides such as glyphosate, glufosinate or bromoxynil (Comai et al., Nature 317:741-744 (1985), Gordon-Kamm et al., Plant Cell 2:603-618 (1990) and Stalker et al., Science 242:419-423 (1988)).

Selectable marker genes for plant transformation that are not of bacterial origin include, for example, mouse dihydrofolate reductase, plant 5-enolpyruvylshikimate-3-phosphate synthase and plant acetolactate synthase (Eichholtz et al., Somatic Cell Mol. Genet. 13:67 (1987), Shah et al., Science 233:478 (1986), Charest et al., Plant Cell Rep. 8:643 (1990)).

Another class of marker genes for plant transformation requires screening of presumptively transformed plant cells rather than direct genetic selection of transformed cells for resistance to a toxic substance such as an antibiotic. These genes are particularly useful to quantify or visualize the spatial pattern of expression of a gene in specific tissues and are frequently referred to as reporter genes because they can be fused to a gene or gene regulatory sequence for the investigation of gene expression. Commonly used genes for screening presumptively transformed cells include α-glucuronidase (GUS), α-galactosidase, luciferase and chloramphenicol, acetyltransferase (Jefferson, R. A., Plant Mol. Biol. Rep. 5:387 (1987), Teeri et al., EMBO J. 8:343 (1989), Koncz et al., Proc. Natl. Acad. Sci U.S.A. 84:131 (1987), DeBlock et al., EMBO J. 3:1681 (1984)).

In vivo methods for visualizing GUS activity that do not require destruction of plant tissues are available (Molecular Probes publication 2908, IMAGENE GREEN, p. 1-4 (1993) and Naleway et al., J. Cell Biol. 115:151a (1991)). However, these in vivo methods for visualizing GUS activity have not proven useful for recovery of transformed cells because of low sensitivity, high fluorescent backgrounds and limitations associated with the use of luciferase genes as selectable markers.

More recently, a gene encoding Green Fluorescent Protein (GFP) has been utilized as a marker for gene expression in prokaryotic and eukaryotic cells (Chalfie et al., Science 263:802 (1994)). GFP and mutants of GFP may be used as screenable markers.

Expression Vectors for Lettuce Transformation: Promoters

Genes included in expression vectors must be driven by a nucleotide sequence comprising a regulatory element, for example, a promoter. Several types of promoters are now well known in the transformation arts, as are other regulatory elements that can be used alone or in combination with promoters.

As used herein, “promoter” includes reference to a region of DNA upstream from the start of transcription and involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A “plant promoter” is a promoter capable of initiating transcription in plant cells. Examples of promoters under developmental control include promoters that preferentially initiate transcription in certain tissues, such as leaves, roots, seeds, fibers, xylem vessels, tracheids, or sclerenchyma. Such promoters are referred to as “tissue-preferred”. Promoters which initiate transcription only in certain tissue are referred to as “tissue-specific”. A “cell type” specific promoter primarily drives expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves. An “inducible” promoter is a promoter which is under environmental control. Examples of environmental conditions that may effect transcription by inducible promoters include anaerobic conditions or the presence of light. Tissue-specific, tissue-preferred, cell type specific, and inducible promoters constitute the class of “non-constitutive” promoters. A “constitutive” promoter is a promoter which is active under most environmental conditions.

A. Inducible Promoters

An inducible promoter is operably linked to a gene for expression in lettuce. Optionally, the inducible promoter is operably linked to a nucleotide sequence encoding a signal sequence which is operably linked to a gene for expression in lettuce. With an inducible promoter the rate of transcription increases in response to an inducing agent.

Any inducible promoter can be used in the instant invention. See Ward et al., Plant Mol. Biol. 22:361-366 (1993). Exemplary inducible promoters include, but are not limited to, that from the ACEI system which responds to copper (Meft et al., PNAS 90:4567-4571 (1993)); In2 gene from maize which responds to benzenesulfonamide herbicide safeners (Hershey et al., Mol. Gen Genetics 227:229-237 (1991) and Gatz et al., Mol. Gen. Genetics 243:32-38 (1994)) or Tet repressor from Tn10 (Gatz et al., Mol. Gen. Genetics 227:229-237 (1991). A particularly preferred inducible promoter is a promoter that responds to an inducing agent to which plants do not normally respond. An exemplary inducible promoter is the inducible promoter from a steroid hormone gene, the transcriptional activity of which is induced by a glucocorticosteroid hormone. Schena et al., Proc. Natl. Acad. Sci. U.S.A. 88:0421 (1991).

B. Constitutive Promoters

A constitutive promoter is operably linked to a gene for expression in lettuce or the constitutive promoter is operably linked to a nucleotide sequence encoding a signal sequence which is operably linked to a gene for expression in lettuce.

Many different constitutive promoters can be utilized in the instant invention. Exemplary constitutive promoters include, but are not limited to, the promoters from plant viruses such as the 35S promoter from CaMV (Odell et al., Nature 313:810-812 (1985) and the promoters from such genes as rice actin (McElroy et al., Plant Cell 2:163-171 (1990)); ubiquitin (Christensen et al., Plant Mol. Biol. 12:619-632 (1989) and Christensen et al., Plant Mol. Biol. 18:675-689 (1992)); pEMU (Last et al., Theor. Appl. Genet. 81:581-588 (1991)); MAS (Velten et al., EMBO J. 3:2723-2730 (1984)) and maize H3 histone (Lepetit et al., Mol. Gen. Genetics 231:276-285 (1992) and Atanassova et al., Plant Journal 2 (3): 291-300 (1992)). The ALS promoter, XbaI/NcoI fragment 5′ to the Brassica napus ALS3 structural gene (or a nucleotide sequence similarity to said XbaI/NcoI fragment), represents a particularly useful constitutive promoter. See PCT application WO 96/30530.

C. Tissue-Specific or Tissue-Preferred Promoters

A tissue-specific promoter is operably linked to a gene for expression in lettuce. Optionally, the tissue-specific promoter is operably linked to a nucleotide sequence encoding a signal sequence which is operably linked to a gene for expression in lettuce. Plants transformed with a gene of interest operably linked to a tissue-specific promoter produce the protein product of the transgene exclusively, or preferentially, in a specific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in the instant invention. Exemplary tissue-specific or tissue-preferred promoters include, but are not limited to, a root-preferred promoter, such as that from the phaseolin gene (Murai et al., Science 23:476-482 (1983) and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci. U.S.A. 82:3320-3324 (1985)); a leaf-specific and light-induced promoter such as that from cab or rubisco (Simpson et al., EMBO J. 4(11):2723-2729 (1985) and Timko et al., Nature 318:579-582 (1985)); an anther-specific promoter such as that from LAT52 (Twell et al., Mol. Gen. Genetics 217:240-245 (1989)); a pollen-specific promoter such as that from Zm13 (Guerrero et al., Mol. Gen. Genetics 244:161-168 (1993)) or a microspore-preferred promoter such as that from apg (Twell et al., Sex. Plant Reprod. 6:217-224 (1993)).

Signal Sequences for Targeting Proteins to Subcellular Compartments

Transport of protein produced by transgenes to a subcellular compartment such as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall or mitochondrion or for secretion into the apoplast, is accomplished by means of operably linking the nucleotide sequence encoding a signal sequence to the 5′ and/or 3′ region of a gene encoding the protein of interest. Targeting sequences at the 5′ and/or 3′ end of the structural gene may determine, during protein synthesis and processing, where the encoded protein is ultimately compartmentalized.

The presence of a signal sequence directs a polypeptide to either an intracellular organelle or subcellular compartment or for secretion to the apoplast. Many signal sequences are known in the art. See, for example Becker et al., Plant Mol. Biol. 20:49 (1992), Close, P. S., Master's Thesis, Iowa State University (1993), Knox, C., et al., “Structure and Organization of Two Divergent Alpha-Amylase Genes from Barley”, Plant Mol. Biol. 9:3-17 (1987), Lerner et al., Plant Physiol. 91:124-129 (1989), Fontes et al., Plant Cell 3:483-496 (1991), Matsuoka et al., Proc. Natl. Acad. Sci. 88:834 (1991), Gould et al., J. Cell. Biol. 108:1657 (1989), Creissen et al., Plant J. 2:129 (1991), Kalderon, et al., A short amino acid sequence able to specify nuclear location, Cell 39:499-509 (1984), Steifel, et al., Expression of a maize cell wall hydroxyproline-rich glycoprotein gene in early leaf and root vascular differentiation, Plant Cell 2:785-793 (1990).

Foreign Protein Genes and Agronomic Genes

With transgenic plants according to the present invention, a foreign protein can be produced in commercial quantities. Thus, techniques for the selection and propagation of transformed plants, which are well understood in the art, yield a plurality of transgenic plants which are harvested in a conventional manner, and a foreign protein then can be extracted from a tissue of interest or from total biomass. Protein extraction from plant biomass can be accomplished by known methods which are discussed, for example, by Heney and Orr, Anal. Biochem. 114:92-6 (1981).

According to a preferred embodiment, the transgenic plant provided for commercial production of foreign protein is lettuce. In another preferred embodiment, the biomass of interest is seed. For the relatively small number of transgenic plants that show higher levels of expression, a genetic map can be generated, primarily via conventional RFLP, PCR and SSR analysis, which identifies the approximate chromosomal location of the integrated DNA molecule. For exemplary methodologies in this regard, see Glick and Thompson, Methods in Plant Molecular Biology and Biotechnology, CRC Press, Boca Raton 269:284 (1993). Map information concerning chromosomal location is useful for proprietary protection of a subject transgenic plant. If unauthorized propagation is undertaken and crosses made with other germplasm, the map of the integration region can be compared to similar maps for suspect plants, to determine if the latter have a common parentage with the subject plant. Map comparisons would involve hybridizations, RFLP, PCR, SSR and sequencing, all of which are conventional techniques.

Likewise, by means of the present invention, agronomic genes can be expressed in transformed plants. More particularly, plants can be genetically engineered to express various phenotypes of agronomic interest. Exemplary genes implicated in this regard include, but are not limited to, those categorized below:

1. Genes that Confer Resistance to Pests or Disease and that Encode

A. Plant disease resistance genes. Plant defenses are often activated by specific interaction between the product of a disease resistance gene (R) in the plant and the product of a corresponding avirulence (Avr) gene in the pathogen. A plant line can be transformed with a cloned resistance gene to engineer plants that are resistant to specific pathogen strains. See, for example Jones et al., Science 266:789 (1994) (cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum); Martin et al., Science 262:1432 (1993) (tomato Pto gene for resistance to Pseudomonas syringae pv. tomato encodes a protein kinase); Mindrinos et al., Cell 78:1089 (1994) (Arabidopsis RSP2 gene for resistance to Pseudomonas syringae).

B. A Bacillus thuringiensis protein, a derivative thereof or a synthetic polypeptide modeled thereon. See, for example, Geiser et al., Gene 48:109 (1986), who disclose the cloning and nucleotide sequence of a Bt δ-endotoxin gene. Moreover, DNA molecules encoding δ-endotoxin genes can be purchased from American Type Culture Collection, Manassas, Va., for example, under ATCC Accession Nos. 40098, 67136, 31995 and 31998.

C. A lectin. See, for example, the disclosure by Van Damme et al., Plant Molec. Biol. 24:25 (1994), who disclose the nucleotide sequences of several Clivia miniata mannose-binding lectin genes.

D. A vitamin-binding protein such as avidin. See PCT application US 93/06487, the contents of which are hereby incorporated by reference. The application teaches the use of avidin and avidin homologues as larvicides against insect pests.

E. An enzyme inhibitor, for example, a protease or proteinase inhibitor or an amylase inhibitor. See, for example, Abe et al., J. Biol. Chem. 262:16793 (1987) (nucleotide sequence of rice cysteine proteinase inhibitor), Huub et al., Plant Molec. Biol. 21:985 (1993) (nucleotide sequence of cDNA encoding tobacco proteinase inhibitor I), Sumitani et al., Biosci. Biotech. Biochem. 57:1243 (1993) (nucleotide sequence of Streptomyces nitrosporeus α-amylase inhibitor).

F. An insect-specific hormone or pheromone such as an ecdysteroid and juvenile hormone, a variant thereof, a mimetic based thereon, or an antagonist or agonist thereof. See, for example, the disclosure by Hammock et al., Nature 344:458 (1990), of baculovirus expression of cloned juvenile hormone esterase, an inactivator of juvenile hormone.

G. An insect-specific peptide or neuropeptide which, upon expression, disrupts the physiology of the affected pest. For example, see the disclosures of Regan, J. Biol. Chem. 269:9 (1994) (expression cloning yields DNA coding for insect diuretic hormone receptor), and Pratt et al., Biochem. Biophys. Res. Comm. 163:1243 (1989) (an allostatin is identified in Diploptera puntata). See also U.S. Pat. No. 5,266,317 to Tomalski et al., who disclose genes encoding insect-specific, paralytic neurotoxins.

H. An insect-specific venom produced in nature by a snake, a wasp, etc. For example, see Pang et al., Gene 116:165 (1992), for disclosure of heterologous expression in plants of a gene coding for a scorpion insectotoxic peptide.

I. An enzyme responsible for a hyper-accumulation of a monoterpene, a sesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivative or another non-protein molecule with insecticidal activity.

J. An enzyme involved in the modification, including the post-translational modification, of a biologically active molecule; for example, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme, a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, a phosphatase, a kinase, a phosphorylase, a polymerase, an elastase, a chitinase and a glucanase, whether natural or synthetic. See PCT application WO 93/02197 in the name of Scott et al., which discloses the nucleotide sequence of a callase gene. DNA molecules which contain chitinase-encoding sequences can be obtained, for example, from the ATCC under Accession Nos. 39637 and 67152. See also Kramer et al., Insect Biochem. Molec. Biol. 23:691 (1993), who teach the nucleotide sequence of a cDNA encoding tobacco hornworm chitinase, and Kawalleck et al., Plant Molec. Biol. 21:673 (1993), who provide the nucleotide sequence of the parsley ubi4-2 polyubiquitin gene.

K. A molecule that stimulates signal transduction. For example, see the disclosure by Botella et al., Plant Molec. Biol. 24:757 (1994), of nucleotide sequences for mung bean calmodulin cDNA clones, and Griess et al., Plant Physiol. 104:1467 (1994), who provide the nucleotide sequence of a maize calmodulin cDNA clone.

L. A hydrophobic moment peptide. See PCT application WO 95/16776 (disclosure of peptide derivatives of tachyplesin which inhibit fungal plant pathogens) and PCT application WO 95/18855 (teaches synthetic antimicrobial peptides that confer disease resistance), the respective contents of which are hereby incorporated by reference.

M. A membrane permease, a channel former or a channel blocker. For example, see the disclosure of Jaynes et al., Plant Sci 89:43 (1993), of heterologous expression of a cecropin-β, lytic peptide analog to render transgenic tobacco plants resistant to Pseudomonas solanacearum.

N. A viral-invasive protein or a complex toxin derived therefrom. For example, the accumulation of viral coat proteins in transformed plant cells imparts resistance to viral infection and/or disease development effected by the virus from which the coat protein gene is derived, as well as by related viruses. See Beachy et al., Ann. rev. Phytopathol. 28:451 (1990). Coat protein-mediated resistance has been conferred upon transformed plants against alfalfa mosaic virus, cucumber mosaic virus, tobacco streak virus, potato virus X, potato virus Y, tobacco etch virus, tobacco rattle virus and tobacco mosaic virus. Id.

O. An insect-specific antibody or an immunotoxin derived therefrom. Thus, an antibody targeted to a critical metabolic function in the insect gut would inactivate an affected enzyme, killing the insect. See Taylor et al., Abstract #497, Seventh Int'l Symposium on Molecular Plant-Microbe Interactions (Edinburgh, Scotland) (1994) (enzymatic inactivation in transgenic tobacco via production of single-chain antibody fragments).

P. A virus-specific antibody. See, for example, Tavladoraki et al., Nature 366:469 (1993), who show that transgenic plants expressing recombinant antibody genes are protected from virus attack.

Q. A developmental-arrestive protein produced in nature by a pathogen or a parasite. Thus, fungal endo-α-1,4-D-polygalacturonases facilitate fungal colonization and plant nutrient release by solubilizing plant cell wall homo-α-1,4-D-galacturonase. See Lamb et al., Bio/Technology 10:1436 (1992). The cloning and characterization of a gene which encodes a bean endopolygalacturonase-inhibiting protein is described by Toubart et al., Plant J. 2:367 (1992).

R. A developmental-arrestive protein produced in nature by a plant. For example, Logemann et al., Bio/Technology 10:305 (1992), have shown that transgenic plants expressing the barley ribosome-inactivating gene have an increased resistance to fungal disease.

S. A lettuce mosaic potyvirus (LMV) coat protein gene introduced into Lactuca sativa in order to increase its resistance to LMV infection. See Dinant et al., Molecular Breeding. 1997, 3: 1, 75-86.

2. Genes that Confer Resistance to an Herbicide

A. An herbicide that inhibits the growing point or meristem, such as an imidazolinone or a sulfonylurea. Exemplary genes in this category code for mutant ALS and AHAS enzyme as described, for example, by Lee et al., EMBO J. 7:1241 (1988), and Miki et al., Theor. Appl Genet. 80:449 (1990), respectively.

B. Glyphosate (resistance conferred by mutant 5-enolpyruvishikimate-3-phosphate synthase (EPSPS) and aroA genes, respectively) and other phosphono compounds such as glufosinate (phosphinothricin acetyl transferase (PAT) and Streptomyces hygroscopicus phosphinothricin-acetyl transferase PAT bar genes), and pyridinoxy or phenoxy proprionic acids and cyclohexones (ACCase inhibitor-encoding genes). See, for example, U.S. Pat. No. 4,940,835 to Shah, et al., which discloses the nucleotide sequence of a form of EPSPS which can confer glyphosate resistance. A DNA molecule encoding a mutant aroA gene can be obtained under ATCC accession number 39256, and the nucleotide sequence of the mutant gene is disclosed in U.S. Pat. No. 4,769,061 to Comai. See also Umaballava-Mobapathie in Transgenic Research. 1999, 8: 1, 33-44 that discloses Lactuca sativa resistant to glufosinate. European patent application No. 0 333 033 to Kumada et al., and U.S. Pat. No. 4,975,374 to Goodman et al., disclose nucleotide sequences of glutamine synthetase genes which confer resistance to herbicides such as L-phosphinothricin. The nucleotide sequence of a phosphinothricin-acetyl-transferase gene is provided in European application No. 0 242 246 to Leemans et al., DeGreef et al., Bio/Technology 7:61 (1989), describe the production of transgenic plants that express chimeric bar genes coding for phosphinothricin acetyl transferase activity. Exemplary of genes conferring resistance to phenoxy proprionic acids and cyclohexones, such as sethoxydim and haloxyfop are the Acc1-S1, Acc1-S2 and Acc1-S3 genes described by Marshall et al., Theor. Appl. Genet. 83:435 (1992).

C. An herbicide that inhibits photosynthesis, such as a triazine (psbA and gs+ genes) and a benzonitrile (nitrilase gene). Przibilla et al., Plant Cell 3:169 (1991), describe the transformation of Chlamydomonas with plasmids encoding mutant psbA genes. Nucleotide sequences for nitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, and DNA molecules containing these genes are available under ATCC Accession Nos. 53435, 67441, and 67442. Cloning and expression of DNA coding for a glutathione S-transferase is described by Hayes et al., Biochem. J. 285:173 (1992).

D. Acetohydroxy acid synthase, which has been found to make plants that express this enzyme resistant to multiple types of herbicides, has been introduced into a variety of plants. See Hattori et al., Mol. Gen. Genet. 246:419, 1995. Other genes that confer tolerance to herbicides include a gene encoding a chimeric protein of rat cytochrome P4507A1 and yeast NADPH-cytochrome P450 oxidoreductase (Shiota et al., Plant Physiol., 106:17, 1994), genes for glutathione reductase and superoxide dismutase (Aono et al., Plant Cell Physiol. 36:1687, 1995), and genes for various phosphotransferases (Datta et al., Plant Mol. Biol. 20:619, 1992).

E. Protoporphyrinogen oxidase (protox) is necessary for the production of chlorophyll, which is necessary for all plant survival. The protox enzyme serves as the target for a variety of herbicidal compounds. These herbicides also inhibit growth of all the different species of plants present, causing their total destruction. The development of plants containing altered protox activity which are resistant to these herbicides are described in U.S. Pat. Nos. 6,288,306; 6,282,837; 5,767,373; and international publication WO 01/12825.

3. Genes that Confer or Contribute to a Value-Added Trait, Such as

A. Increased iron content of the lettuce, for example by transforming a plant with a soybean ferritin gene as described in Goto et al., Acta Horticulturae. 2000, 521, 101-109.

B. Decreased nitrate content of leaves, for example by transforming a lettuce with a gene coding for a nitrate reductase. See for example Curtis et al., Plant Cell Report. 1999, 18: 11, 889-896.

C. Increased sweetness of the lettuce by transferring a gene coding for monellin that elicits a flavor 100,000 times sweeter than sugar on a molar basis. See Penarrubia et al., Biotechnology. 1992, 10: 561-564.

D. Modified fatty acid metabolism, for example, by transforming a plant with an antisense gene of stearyl-ACP desaturase to increase stearic acid content of the plant. See Knultzon et al., Proc. Natl. Acad. Sci. USA 89:2625 (1992).

E. Modified carbohydrate composition effected, for example, by transforming plants with a gene coding for an enzyme that alters the branching pattern of starch. See Shiroza et al., J. Bacteriol. 170:810 (1988) (nucleotide sequence of Streptococcus mutants fructosyltransferase gene), Steinmetz et al., Mol. Gen. Genet. 20:220 (1985) (nucleotide sequence of Bacillus subtilis levansucrase gene), Pen et al., Bio/Technology 10:292 (1992) (production of transgenic plants that express Bacillus lichenifonnis α-amylase), Elliot et al., Plant Molec. Biol. 21:515 (1993) (nucleotide sequences of tomato invertase genes), Søgaard et al., J. Biol. Chem. 268:22480 (1993) (site-directed mutagenesis of barley α-amylase gene), and Fisher et al., Plant Physiol. 102:1045 (1993) (maize endosperm starch branching enzyme II).

4. Genes that Control Male-Sterility

A. Introduction of a deacetylase gene under the control of a tapetum-specific promoter and with the application of the chemical N-Ac-PPT. See international publication WO 01/29237.

B. Introduction of various stamen-specific promoters. See international publications WO 92/13956 and WO 92/13957.

C. Introduction of the barnase and the barstar genes. See Paul et al., Plant Mol. Biol. 19:611-622, 1992).

Methods for Lettuce Transformation

Numerous methods for plant transformation have been developed, including biological and physical, plant transformation protocols. See, for example, Miki et al., “Procedures for Introducing Foreign DNA into Plants” in Methods in Plant Molecular Biology and Biotechnology, Glick B.R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages 67-88. In addition, expression vectors and in vitro culture methods for plant cell or tissue transformation and regeneration of plants are available. See, for example, Gruber et al., “Vectors for Plant Transformation” in Methods in Plant Molecular Biology and Biotechnology, Glick B.R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages 89-119.

A. Agrobacterium-Mediated Transformation

One method for introducing an expression vector into plants is based on the natural transformation system of Agrobacterium. See, for example, Horsch et al., Science 227:1229 (1985), Curtis et al., Journal of Experimental Botany. 1994, 45: 279, 1441-1449, Torres et al., Plant cell Tissue and Organic Culture. 1993, 34: 3, 279-285, Dinant et al., Molecular Breeding. 1997, 3: 1, 75-86. A. tumefaciens and A. rhizogenes are plant pathogenic soil bacteria which genetically transform plant cells. The Ti and Ri plasmids of A. tumefaciens and A. rhizogenes, respectively, carry genes responsible for genetic transformation of the plant. See, for example, Kado, C. I., Crit. Rev. Plant Sci. 10:1 (1991). Descriptions of Agrobacterium vector systems and methods for Agrobacterium-mediated gene transfer are provided by Gruber et al., supra, Miki et al., supra, and Moloney et al., Plant Cell Reports 8:238 (1989). See also, U.S. Pat. No. 5,591,616 issued Jan. 7, 1997.

B. Direct Gene Transfer

Several methods of plant transformation collectively referred to as direct gene transfer have been developed as an alternative to Agrobacterium-mediated transformation. A generally applicable method of plant transformation is microprojectile-mediated transformation wherein DNA is carried on the surface of microprojectiles measuring 1 to 4 μm. The expression vector is introduced into plant tissues with a biolistic device that accelerates the microprojectiles to speeds of 300 to 600 m/s which is sufficient to penetrate plant cell walls and membranes. Russell, D. R., et al. Pl. Cell. Rep. 12(3, January), 165-169 (1993), Aragao, F. J. L., et al. Plant Mol. Biol. 20(2, October), 357-359 (1992), Aragao, F. J. L., et al. Pl. Cell. Rep. 12(9, July), 483-490 (1993). Aragao, Theor. Appl. Genet. 93:142-150 (1996), Kim, J.; Minamikawa, T. Plant Science 117: 131-138 (1996), Sanford et al., Part. Sci. Technol. 5:27 (1987), Sanford, J. C., Trends Biotech. 6:299 (1988), Klein et al., Bio/Technology 6:559-563 (1988), Sanford, J. C., Physiol Plant 7:206 (1990), Klein et al., Biotechnology 10:268 (1992).

Another method for physical delivery of DNA to plants is sonication of target cells. Zhang et al., Bio/Technology 9:996 (1991). Alternatively, liposome and spheroplast fusion have been used to introduce expression vectors into plants. Deshayes et al., EMBO J., 4:2731 (1985), Christou et al., Proc Natl. Acad. Sci. U.S.A. 84:3962 (1987). Direct uptake of DNA into protoplasts using CaCl₂ precipitation, polyvinyl alcohol or poly-L-ornithine has also been reported. Hain et al., Mol. Gen. Genet. 199:161 (1985) and Draper et al., Plant Cell Physiol. 23:451 (1982). Electroporation of protoplasts and whole cells and tissues have also been described. Saker, M.; Kuhne, T. Biologia Plantarum 40(4): 507-514 (1997/98), Donn et al., In Abstracts of VIIth International Congress on Plant Cell and Tissue Culture IAPTC, A2-38, p 53 (1990); D'Halluin et al., Plant Cell 4:1495-1505 (1992) and Spencer et al., Plant Mol. Biol. 24:51-61 (1994). See also Chupean et al., Biotechnology. 1989, 7: 5, 503-508.

Following transformation of lettuce target tissues, expression of the above-described selectable marker genes allows for preferential selection of transformed cells, tissues and/or plants, using regeneration and selection methods now well known in the art.

The foregoing methods for transformation would typically be used for producing a transgenic line. The transgenic line could then be crossed, with another (non-transformed or transformed) line, in order to produce a new transgenic lettuce line. Alternatively, a genetic trait which has been engineered into a particular lettuce cultivar using the foregoing transformation techniques could be moved into another line using traditional backcrossing techniques that are well known in the plant breeding arts. For example, a backcrossing approach could be used to move an engineered trait from a public, non-elite inbred line into an elite inbred line, or from an inbred line containing a foreign gene in its genome into an inbred line or lines which do not contain that gene. As used herein, “crossing” can refer to a simple X by Y cross, or the process of backcrossing, depending on the context.

Single-Gene Conversions

When the terms lettuce plant, cultivar or lettuce line are used in the context of the present invention, this also includes any single gene conversions of that line. The term “single gene converted plant” as used herein refers to those lettuce plants which are developed by a plant breeding technique called backcrossing wherein essentially all of the desired morphological and physiological characteristics of a cultivar are recovered in addition to the single gene transferred into the line via the backcrossing technique. Backcrossing methods can be used with the present invention to improve or introduce a characteristic into the line. The term “backcrossing” as used herein refers to the repeated crossing of a hybrid progeny back to one of the parental lettuce plants for that line, backcrossing 1, 2, 3, 4, 5, 6, 7, 8 or more times to the recurrent parent. The parental lettuce plant which contributes the gene for the desired characteristic is termed the nonrecurrent or donor parent. This terminology refers to the fact that the nonrecurrent parent is used one time in the backcross protocol and therefore does not recur. The parental lettuce plant to which the gene or genes from the nonrecurrent parent are transferred is known as the recurrent parent as it is used for several rounds in the backcrossing protocol (Poehlman & Sleper, 1994; Fehr, 1987). In a typical backcross protocol, the original cultivar of interest (recurrent parent) is crossed to a second line (nonrecurrent parent) that carries the single gene of interest to be transferred. The resulting progeny from this cross are then crossed again to the recurrent parent and the process is repeated until a lettuce plant is obtained wherein essentially all of the desired morphological and physiological characteristics of the recurrent parent are recovered in the converted plant, in addition to the single transferred gene from the nonrecurrent parent.

The selection of a suitable recurrent parent is an important step for a successful backcrossing procedure. The goal of a backcross protocol is to alter or substitute a single trait or characteristic in the original line. To accomplish this, a single gene of the recurrent cultivar is modified or substituted with the desired gene from the nonrecurrent parent, while retaining essentially all of the rest of the desired genetic, and therefore the desired physiological and morphological, constitution of the original line. The choice of the particular nonrecurrent parent will depend on the purpose of the backcross, one of the major purposes is to add some commercially desirable, agronomically important trait to the plant. The exact backcrossing protocol will depend on the characteristic or trait being altered to determine an appropriate testing protocol. Although backcrossing methods are simplified when the characteristic being transferred is a dominant allele, a recessive allele may also be transferred. In this instance it may be necessary to introduce a test of the progeny to determine if the desired characteristic has been successfully transferred.

Many single gene traits have been identified that are not regularly selected for in the development of a new line but that can be improved by backcrossing techniques. Single gene traits may or may not be transgenic, examples of these traits include but are not limited to, male sterility, modified fatty acid metabolism, modified carbohydrate metabolism, herbicide resistance, resistance for bacterial, fungal, or viral disease, insect resistance, enhanced nutritional quality, industrial usage, yield stability and yield enhancement. These genes are generally inherited through the nucleus. Several of these single gene traits are described in U.S. Pat. Nos. 5,777,196, 5,948,957 and 5,969,212, the disclosures of which are specifically hereby incorporated by reference.

Tissue Culture

Further reproduction of the variety can occur by tissue culture and regeneration. Tissue culture of various tissues of lettuce and regeneration of plants therefrom is well known and widely published. For example, reference may be had to Teng et al., HortScience. 1992, 27: 9, 1030-1032 Teng et al., HortScience. 1993, 28: 6, 669-1671, Zhang et al., Journal of Genetics and Breeding. 1992, 46: 3, 287-290, Webb et al., Plant Cell Tissue and Organ Culture. 1994, 38: 1, 77-79, Curtis et al., Journal of Experimental Botany. 1994, 45: 279, 1441-1449, Nagata et al., Journal for the American Society for Horticultural Science. 2000, 125: 6, 669-672, and Ibrahim et al., Plant Cell, Tissue and Organ Culture. (1992), 28(2): 139-145. It is clear from the literature that the state of the art is such that these methods of obtaining plants are routinely used and have a very high rate of success. Thus, another aspect of this invention is to provide cells which upon growth and differentiation produce lettuce plants having the physiological and morphological characteristics of variety Valley Fresh.

As used herein, the term “tissue culture” indicates a composition comprising isolated cells of the same or a different type or a collection of such cells organized into parts of a plant. Exemplary types of tissue cultures are protoplasts, calli, meristematic cells, and plant cells that can generate tissue culture that are intact in plants or parts of plants, such as leaves, pollen, embryos, roots, root tips, anthers, pistils, flowers, seeds, petioles, suckers and the like. Means for preparing and maintaining plant tissue culture are well known in the art. By way of example, a tissue culture comprising organs has been used to produce regenerated plants. U.S. Pat. Nos. 5,959,185; 5,973,234 and 5,977,445 describe certain techniques, the disclosures of which are incorporated herein by reference.

Additional Breeding Methods

This invention also is directed to methods for producing a lettuce plant by crossing a first parent lettuce plant with a second parent lettuce plant wherein the first or second parent lettuce plant is a lettuce plant of cultivar Valley Fresh. Further, both first and second parent lettuce plants can come from lettuce cultivar Valley Fresh. Thus, any such methods using lettuce cultivar Valley Fresh are part of this invention: selfing, backcrosses, hybrid production, crosses to populations, and the like. All plants produced using lettuce cultivar Valley Fresh as at least one parent are within the scope of this invention, including those developed from cultivars derived from lettuce cultivar Valley Fresh. Advantageously, this lettuce cultivar could be used in crosses with other, different, lettuce plants to produce the first generation (F₁) lettuce hybrid seeds and plants with superior characteristics. The cultivar of the invention can also be used for transformation where exogenous genes are introduced and expressed by the cultivar of the invention. Genetic variants created either through traditional breeding methods using lettuce cultivar Valley Fresh or through transformation of cultivar Valley Fresh by any of a number of protocols known to those of skill in the art are intended to be within the scope of this invention.

The following describes breeding methods that may be used with lettuce cultivar Valley Fresh in the development of further lettuce plants. One such embodiment is a method for developing cultivar Valley Fresh progeny lettuce plants in a lettuce plant breeding program comprising: obtaining the lettuce plant, or a part thereof, of cultivar Valley Fresh, utilizing said plant or plant part as a source of breeding material, and selecting a lettuce cultivar Valley Fresh progeny plant with molecular markers in common with cultivar Valley Fresh and/or with morphological and/or physiological characteristics selected from the characteristics listed in Table 1. Breeding steps that may be used in the lettuce plant breeding program include pedigree breeding, backcrossing, mutation breeding, and recurrent selection. In conjunction with these steps, techniques such as RFLP-enhanced selection, genetic marker enhanced selection (for example SSR markers) and the making of double haploids may be utilized.

Another method involves producing a population of lettuce cultivar Valley Fresh progeny lettuce plants, comprising crossing cultivar Valley Fresh with another lettuce plant, thereby producing a population of lettuce plants, which, on average, derive 50% of their alleles from lettuce cultivar Valley Fresh. A plant of this population may be selected and repeatedly selfed or sibbed with a lettuce cultivar resulting from these successive filial generations. One embodiment of this invention is the lettuce cultivar produced by this method and that has obtained at least 50% of its alleles from lettuce cultivar Valley Fresh.

One of ordinary skill in the art of plant breeding would know how to evaluate the traits of two plant varieties to determine if there is no significant difference between the two traits expressed by those varieties. For example, see Fehr and Walt, Principles of Cultivar Development, p 261-286 (1987). Thus the invention includes lettuce cultivar Valley Fresh progeny lettuce plants comprising a combination of at least two cultivar Valley Fresh traits selected from the group consisting of those listed in Table 1 or the cultivar Valley Fresh combination of traits listed in the Summary of the Invention, so that said progeny lettuce plant is not significantly different for said traits than lettuce cultivar Valley Fresh as determined at the 5% significance level when grown in the same environmental conditions. Using techniques described herein, molecular markers may be used to identify said progeny plant as a lettuce cultivar Valley Fresh progeny plant. Mean trait values may be used to determine whether trait differences are significant, and preferably the traits are measured on plants grown under the same environmental conditions. Once such a variety is developed its value is substantial since it is important to advance the germplasm base as a whole in order to maintain or improve traits such as yield, disease resistance, pest resistance, and plant performance in extreme environmental conditions.

Progeny of lettuce cultivar Valley Fresh may also be characterized through their filial relationship with lettuce cultivar Valley Fresh, as for example, being within a certain number of breeding crosses of lettuce cultivar Valley Fresh. A breeding cross is a cross made to introduce new genetics into the progeny, and is distinguished from a cross, such as a self or a sib cross, made to select among existing genetic alleles. The lower the number of breeding crosses in the pedigree, the closer the relationship between lettuce cultivar Valley Fresh and its progeny. For example, progeny produced by the methods described herein may be within 1, 2, 3, 4 or 5 breeding crosses of lettuce cultivar Valley Fresh.

As used herein, the term “plant” includes plant cells, plant protoplasts, plant cell tissue cultures from which lettuce plants can be regenerated, plant calli, plant clumps and plant cells that are intact in plants or parts of plants, such as leaves, pollen, embryos, cotyledons, hypocotyl, roots, root tips, anthers, pistils, flowers, seeds, stems and the like.

TABLES

In the tables that follow, the traits and characteristics of lettuce cultivar Valley Fresh are given compared to two commercial romaine lettuce cultivars, Green Thunder and PIC Cos.

Table 2 below shows the cotyledon length for Valley Fresh as compared to the cotyledon length for Green Thunder and PIC Cos. Cotyledon length is measured in millimeters on 20 day old seedlings. An analysis of variance was performed on the data and is shown below the data. As can be seen from the data in Table 2, Valley Fresh has a significantly longer cotyledon leaf length than Green Thunder and PIC Cos.

TABLE 2 Cotyledon length VALLEY Green (mm) FRESH Thunder PIC Cos 25 15 17 25 16 18 25 14 19 23 17 18 20 18 19 20 16 20 23 19 15 22 17 18 20 17 19 20 17 16 22 19 16 20 16 20 22 17 20 20 17 17 23 17 20 22 15 15 23 16 17 Anova: Single Factor SUMMARY Groups Count Sum Average Variance VALLEY FRESH 17 375 22.058824 3.433824 Green Thunder 17 283 16.647059 1.742647 PIC Cos 17 304 17.882353 2.985294 ANOVA Source of Variation SS df MS F P-value F crit Between Varieties 273.45098  2 136.72549 50.25586 1.69E−12 3.190721 Within Varieties 130.58824 48 2.7205882 Total 404.03922 50

Table 3 below shows the cotylendon width for Valley Fresh as compared to the cotylendon width for Green Thunder and PIC Cos. Cotyledon width is measured in millimeters on 20 day old seedlings. An analysis of variance was performed on the data and is shown below the data. As can be seen from the data in Table 3, Valley Fresh has a significantly wider cotyledon leaf width than Green Thunder and PIC Cos.

TABLE 3 Cotyledon width Valley Green (mm) Fresh Thunder PIC Cos 11 9 8 12 9 8 12 8 7 12 10 7 10 9 7 11 10 7 12 9 5 11 8 7 11 10 8 11 9 7 11 9 7 10 8 9 11 10 8 12 10 6 11 8 9 12 8 6 12 10 8 Anova: Single Factor SUMMARY Groups Count Sum Average Variance VALLEY FRESH 17 375 22.058824 3.433824 Green Thunder 17 283 16.647059 1.742647 PIC Cos 17 304 17.882353 2.985294 ANOVA Source of Variation SS df MS F P-value F crit Between Varieties 273.45098  2 136.72549 50.25586 1.69E−12 3.190721 Within Varieties 130.58824 48 2.7205882 Total 404.03922 50

Table 4 below shows the cotyledon index for Valley Fresh as compared to the cotyledon index for Green Thunder and PIC Cos. Cotyledon index is calculated by dividing the cotyledon leaf length by the cotyledon leaf width on 20 day old seedlings. An analysis of variance was performed on the data and is shown below the data. As can be seen from the data in Table 4, Valley Fresh has a significantly different cotyledon leaf index than Green Thunder and PIC Cos.

TABLE 4 Valley Green Cotyledon index Fresh Thunder PIC Cos 2.27 1.70 2.10 2.08 1.80 2.20 2.08 1.80 2.70 1.92 1.70 2.60 2.00 2.00 2.70 1.82 1.60 2.90 1.92 2.10 3.00 2.00 2.10 2.60 1.82 1.70 2.40 1.82 1.90 2.30 2.00 2.10 2.30 2.00 2.00 2.20 2.00 1.70 2.50 1.67 1.70 2.80 2.09 2.10 2.20 1.83 1.90 2.50 1.92 1.60 2.10 Anova: Single Factor SUMMARY Groups Count Sum Average Variance VALLEY FRESH 17 33.23484848 1.9549911 0.020062 Green Thunder 17 31.5 1.8529412 0.033897 PIC Cos 17 42.1 2.4764706 0.079412 ANOVA Source of Variation SS df MS F P-value F crit Between Varieties 3.8031492  2 1.9015746 42.77343 2.16E−11 3.190721 Within Varieties 2.1339318 48 0.0444569 Total 5.9370811 50

Table 5 below shows the fourth leaf length for Valley Fresh as compared to the fourth leaf length for Green Thunder and PIC Cos. Fourth leaf length is measured in centimeters on 20 day old seedlings. An analysis of variance was performed on the data and is shown below the data. As can be seen from the data in Table 5, Valley Fresh has a significantly longer fourth leaf length than Green Thunder and PIC Cos.

TABLE 5 4th Leaf Length (cm) Valley Fresh Green Thunder PIC Cos 5.2 8.0 6.50 9.2 7.3 6.00 7.5 8.0 3.80 7.0 7.5 6.80 8.5 6.5 6.60 5.4 6.6 5.80 6.2 7.0 6.20 6.5 6.8 6.40 5.4 4.2 6.50 9.0 5.2 6.00 5.0 6.0 6.00 8.3 7.0 7.00 8.0 5.0 6.50 7.5 6.0 6.60 7.8 7.2 6.00 9.0 3.5 6.50 Anova: Single Factor SUMMARY Groups Count Sum Average Variance VALLEY FRESH 16 115.5 7.21875 2.093625 Green Thunder 16 101.8 6.3625 1.7105 PIC Cos 16 99.2 6.2 0.52 ANOVA Source of Variation SS df MS F P-value F crit Between Varieties 9.58625 2 4.793125 3.325384 0.044984 3.20432 Within Varieties 64.86188 45 1.441375 Total 74.44812 47

Table 6 below shows the fourth leaf index for Valley Fresh as compared to the fourth leaf index for Green Thunder and PIC Cos. Fourth leaf index is calculated by dividing the fourth leaf length by the fourth leaf width. An analysis of variance was performed on the data and is shown below the data. As can be seen from the data in Table 6, Valley Fresh has a significantly different fourth leaf index than Green Thunder and PIC Cos.

TABLE 6 4th Leaf Index Valley Fresh Green Thunder PIC Cos 2.08 2.29 2.03 2.30 2.09 2.14 2.14 2.29 1.90 2.19 2.08 2.13 2.43 2.32 2.20 2.16 2.20 1.93 2.07 2.33 2.07 2.32 2.43 2.13 2.25 2.00 2.03 2.25 2.00 2.14 2.00 2.00 2.00 2.08 2.06 2.00 2.29 2.27 2.03 2.21 2.00 2.13 2.44 2.06 2.00 2.25 1.17 1.86 Anova: Single Factor SUMMARY Groups Count Sum Average Variance VALLEY FRESH 16 35.44112045 2.21507 0.015831 Green Thunder 16 33.57916985 2.098698 0.082356 PIC Cos 16 32.72397273 2.045248 0.009356 ANOVA Source of Variation SS df MS F P-value F crit Between Varieties 0.241273  2 0.120637 3.365243 0.043449 3.20432 Within Varieties 1.613151 45 0.035848 Total 1.854425 47

Tables 7 through 14 show data collected at twelve different locations in California and Arizona.

Table 7 below shows the plant weight in grams at harvest maturity of Valley Fresh as compared to Green Thunder and PIC Cos at harvest maturity. An analysis of variance was performed on the data and is shown below the data. As can be seen from the data in Table 7, Valley Fresh is significantly heavier in plant weight at harvest maturity than either Green Thunder or PIC Cos.

TABLE 7 Plant Weight in grams Trail Location: Loc. 1 Loc. 2 Loc. 3 Loc. 4 Loc. 5 Loc. 6 Loc. 7 Loc. 8 Loc. 9 Loc. 10 Loc. 11 Loc. 12 VALLEY FRESH 640 900 780 970 1170 810 600 710 490 760 610 600 860 950 870 860 810 1050 680 610 610 640 400 660 920 780 830 710 900 1380 780 710 670 690 560 500 1060 950 930 770 1110 1020 660 720 610 700 720 620 710 700 1040 890 760 860 740 580 670 700 570 520 970 890 930 790 820 800 770 680 820 630 630 620 810 890 910 710 910 810 670 660 700 680 640 530 810 930 920 980 970 940 620 680 700 660 580 560 900 830 840 940 1020 1050 740 720 780 600 510 600 900 850 950 920 880 1160 650 680 730 510 930 590 Green Thunder 560 580 950 700 880 940 730 730 570 570 670 660 500 810 850 800 580 990 730 560 560 750 840 670 580 860 730 780 1060 750 710 650 790 620 720 730 730 810 950 760 910 940 680 800 740 530 560 660 720 580 900 960 750 750 600 650 530 510 710 630 880 750 710 880 1040 690 520 630 600 650 700 690 920 580 630 950 850 560 600 570 780 540 560 840 780 680 950 700 890 950 550 630 590 680 860 700 720 750 790 710 1170 620 700 650 650 640 720 650 940 750 700 750 1040 1070 630 600 630 570 630 680 PIC Cos 780 880 530 920 750 880 620 550 620 560 550 860 670 850 810 810 850 630 740 660 620 540 820 710 750 600 600 910 440 790 590 640 650 680 670 660 790 750 860 800 930 450 720 710 570 530 650 690 910 740 760 920 800 1030 640 630 610 670 580 740 720 830 860 840 870 620 700 660 490 620 610 860 740 870 1030 670 830 990 540 600 710 690 680 650 730 870 750 850 980 660 600 670 610 650 580 610 600 680 890 730 610 920 610 640 700 570 680 590 690 940 910 800 990 810 530 610 540 600 710 800 SUMMARY Loc. 1 Loc. 2 Loc. 3 Loc. 4 Loc. 5 Loc. 6 Loc. 7 Loc. 8 Loc. 9 Loc. 10 Loc. 11 Loc. 12 Anova: Two-Factor With Replication VALLEY FRESH Count 10 10 10 10 10 10 10 10 10 10 10 10 Sum 8580 8670 9000 8540 9350 9880 6910 6750 6780 6570 6150 5800 Average 858 867 900 854 935 988 691 675 678 657 615 580 Variance 14973.33 6334.444 5355.5556 10560 17627.78 34373.33 3943.33 2227.78 8773.3 4645.56 19405.56 2600 Green Thunder Count 10 10 10 10 10 10 10 10 10 10 10 10 Sum 7330 7150 8160 7990 9170 8260 6450 6470 6440 6060 6970 6910.00 Average 733 715 816 799 917 826 645 647 644 606 697 691.00 Variance 23290 10961.11 14382.222 9676.67 29201.11 30115.56 5761.11 5178.89 8848.9 5715.56 10112.22 3521.11 PIC Cos Count 10 10 10 10 10 10 10 10 10 10 10 10 Sum 7380 8010 8000 8250 8050 7780 6290 6370 6120 6110 6530 7170 Average 738 801 800 825 805 778 629 637 612 611 653 717 Variance 6728.889 11076.67 21933.333 6738.89 29072.22 34284.44 5143.33 1912.22 4528.9 3565.56 6223.333 9378.89 TOTALS VALLEY FRESH Green Thunder PIC COS Count 120 120 120 Sum 92980 87360 86060 Average 774.8333 728 717.1667 Variance 27726.02 19925.38 17188.54 ANOVA Variety 225446.7 2 112723.33 9.47761 1E−04 3.023601 Locations 3251633 11 295603.03 24.8539 4.99E−37 1.818261 Interaction 610780 22 27762.727 2.33425 0.000772 1.575174 Within 3853540 324 11893.642 Total 103469.60 359

Table 8 below shows the plant height of Valley Fresh at harvest maturity as compared to Green Thunder and PIC Cos at harvest maturity. Plant height is measured in centimeters. An analysis of variance was performed on the data and is shown below the data. As can be seen from the data in Table 8, Valley Fresh is significantly taller than Green Thunder.

TABLE 8 Plant Height (cm) Trail Location: Loc. 1 Loc. 2 Loc. 3 Loc. 4 Loc. 5 Loc. 6 Loc. 7 Loc. 8 Loc. 9 Loc. 10 Loc. 11 Loc. 12 VALLEY FRESH 35 34 36 34 38 40 35 35 37 38 34 36 34 37 37 35 34 37 34 33 39 36 34 35 37 35 36 32 39 37 35 36 40 36 35 35 35 33 36 35 39 38 34 34 38 37 36 35 34 34 36 34 36 40 34 35 39 34 35 35 37 36 38 34 37 40 34 35 39 37 34 36 33 37 34 34 38 36 36 35 38 35 36 36 34 36 34 36 40 39 34 35 36 35 35 36 33 35 35 38 37 39 35 34 37 37 34 35 35 37 38 36 36 41 34 35 39 36 34 35 Green Thunder 28 30 33 37 39 35 33 33 35 37 35 35 32 31 37 35 38 36 32 34 32 36 35 34 30 31 34 34 39 36 33 33 36 35 33 35 32 32 39 33 36 36 33 34 37 35 33 35 30 31 37 38 39 32 34 33 34 35 33 35 29 30 34 36 39 35 33 34 34 38 34 36 31 30 35 34 38 35 33 35 38 35 34 36 30 30 37 37 37 38 33 34 38 35 35 35 30 33 38 37 33 31 35 34 34 35 34 34 33 31 33 32 34 34 32 34 34 36 35 34 PIC Cos 32 31 34 33 37 34 37 35 39 39 36 37 34 31 34 34 35 35 36 33 38 40 38 35 32 31 34 35 34 32 36 35 39 40 36 36 30 30 36 36 34 38 36 36 40 38 37 37 31 33 33 35 37 37 36 35 40 40 35 35 30 33 38 34 38 37 33 37 39 40 36 35 30 32 36 35 36 30 34 37 40 39 37 35 30 33 35 33 36 36 34 36 38 40 35 35 31 33 34 33 36 34 36 36 40 39 38 36 32 34 36 35 35 35 34 35 38 39 36 37 Anova: Two-Factor With Replication Plant Height (cm) SUMMARY Loc. 1 Loc. 2 Loc. 3 Loc. 4 Loc. 5 Loc. 6 Loc. 7 Loc. 8 Loc. 9 Loc. 10 Loc. 11 Loc. 12 VALLEY FRESH Count 10 10 10 10 10 10 10 10 10 10 10 10 Sum 347 354 360 348 374 387 345 347 382 361 347 354 Average 34.7 35.4 36 34.8 37.4 38.7 34.5 34.7 38.2 36.1 34.7 35.4 Variance 2.011111 2.044444 2 2.62222 3.155556 2.677778 0.5 0.67778 1.5111 1.43333 0.677778 0.26667 Green Thunder Count 10 10 10 10 10 10 10 10 10 10 10 10 Sum 305 309 357 353 372 348 331 338 352 357 341 349 Average 30.5 30.9 35.7 35.3 37.2 34.8 33.1 33.8 35.2 35.7 34.1 34.9 Variance 2.277778 0.988889 4.6777778 4.01111 4.844444 4.177778 0.76667 0.4 3.9556 1.12222 0.766667 0.54444 PIC Cos Count 10 10 10 10 10 10 10 10 10 10 10 10 Sum 312 321 350 343 358 348 352 355 391 394 364 358 Average 31.2 32.1 35 34.3 35.8 34.8 35.2 3.5 39.1 39.4 36.4 35.8 Variance 1.733333 1.655556 2.2222222 1.12222 1.733333 5.955556 1.73333 1.38889 0.7667 0.48889 1.155556 0.84444 TOTALS VALLEY FRESH Green Thunder PIC COS Count 120 120 120 Sum 4306 4112 4246 Average 35.88333 34.26667 35.38333 Variance 3.448459 5.726611 6.72577 ANOVA Source of Variation SS df MS F P-value F crit Variety 164.4222 2 82.211111 42.9481 2.85E−17 3.023601 Locations 863.9556 11 78.541414 41.031 6.58E−55 1.818261 Interaction 408.0444 22 18.547475 9.68943 1.38E−24 1.575174 Within 620.2 324 1.9141975 Total 2056.622 359

Table 9 below shows the frame leaf length of Valley Fresh at harvest maturity as compared to Green Thunder and PIC Cos at harvest maturity. Frame leaf length is measured in centimeters. An analysis of variance was performed on the data and is shown below the data. As can be seen in Table 9, Valley Fresh has a significantly different frame leaf length Green Thunder.

TABLE 9 Frame Leaf Length (cm) at Harvest Maturity Trail Location: Loc. 1 Loc. 2 Loc. 3 Loc. 4 Loc. 5 Loc. 6 Loc. 7 Loc. 8 Loc. 9 Loc. 10 Loc. 11 Loc. 12 VALLEY FRESH 32 33 30 29 38 34 36 33 35 37 31 32 30 32 27 30 35 32 34 36 34 35 35 35 32 31 32 28 34 37 34 34 37 34 34 37 29 28 27 30 33 36 33 34 36 36 34 32 30 31 28 28 38 36 36 35 34 36 35 34 33 29 31 31 32 35 35 36 36 34 34 36 33 31 30 27 36 32 34 35 34 34 37 34 31 31 28 30 36 37 36 34 35 35 34 32 29 33 27 30 34 36 33 34 37 36 34 38 30 31 29 30 34 38 34 33 35 36 35 34 Green Thunder 28 25 30 30 33 33 32 33 35 35 30 33 26 26 33 30 32 32 33 33 35 35 33 32 26 26 32 30 31 36 31 31 34 36 32 32 27 24 30 32 32 33 33 33 36 36 31 31 26 28 31 30 35 35 32 33 34 36 32 32 26 27 35 32 33 36 30 32 34 36 32 30 27 29 33 33 34 32 31 34 35 34 32 31 25 29 33 33 30 33 33 33 36 34 31 31 26 26 31 31 34 32 33 32 35 34 32 33 28 29 33 34 35 34 32 32 35 35 33 34 PIC Cos 28 30 31 32 34 33 36 36 39 39 35 34 29 30 31 31 36 33 34 34 39 40 32 32 30 28 31 30 32 34 35 33 40 38 35 35 27 29 29 33 30 32 33 35 40 40 35 34 30 28 30 33 36 35 36 34 41 39 31 34 30 28 28 31 34 31 33 34 38 40 33 35 29 35 29 30 31 32 35 35 40 39 35 36 30 29 31 30 33 36 35 36 40 39 38 35 32 28 31 28 33 34 36 33 41 38 34 36 27 30 30 30 33 34 33 33 39 40 36 35 Anova: Two-Factor With Replication Frame leaf length (cm) SUMMARY Loc. 1 Loc. 2 Loc. 3 Loc. 4 Loc. 5 Loc. 6 Loc. 7 Loc. 8 Loc. 9 Loc. 10 Loc. 11 Loc. 12 VALLEY FRESH Count 10 10 10 10 10 10 10 10 10 10 10 10 Sum 309 310 289 293 350 353 345 344 353 353 343 344 Average 30.9 31 28.9 29.3 35 35.3 34.5 34.4 35.3 35.3 34.3 34.4 Variance 2.322222 2.444444 3.2111111 1.56667 4 4.233333 1.38889 1.15556 1.3444 1.12222 2.233333 4.48889 Green Thunder Count 10 10 10 10 10 10 10 10 10 10 10 10.00 Sum 265 269 321 315 329 336 320 326 349 351 318 16.33 Average 26.5 26.9 32.1 31.5 32.9 33.6 32 32.6 34.9 35.1 31.8 1.63 Variance 0.944444 3.211111 2.5444444 2.27778 2.766667 2.488889 1.11111 0.71111 0.5444 0.76667 0.844444 0.01 PIC Cos Count 10 10 10 10 10 10 10 10 10 10 10 10.00 Sum 292 295 301 308 332 334 346 343 397 392 344 17.07 Average 29.2 29.5 30.1 30.8 33.2 33.4 34.6 34.3 39.7 39.2 34.4 1.71 Variance 2.4 4.5 1.2111111 2.4 3.733333 2.266667 1.6 1.34444 0.9 0.62222 4.044444 0.02 TOTALS VALLEY FRESH Green Thunder PIC COS Count 120 120 120 Sum 3986 3818 4030 Average 33.21667 31.81667 33.58333 Variance 7.784594 8.033333 12.76611 ANOVA Source of Variation SS df MS F P-value F crit Variety 208.6222 2 104.31111 49.7012 1.49E−19 3.023601 Locations 2219.189 11 201.74444 96.1253 5.2E−95 1.818261 Interaction 502.3111 22 22.832323 10.8789 1.21E−27 1.575174 Within 680 324 2.0987654 Total 3610.122 359

Table 10 below shows the frame leaf width of Valley Fresh at harvest maturity as compared to Green Thunder and PIC Cos at harvest maturity. Frame leaf width is measured in centimeters. An analysis of variance was performed on the data and is shown below the data. As can be seen in Table 10, Valley Fresh has a significantly different frame leaf width than Green Thunder and PIC Cos at harvest maturity.

TABLE 10 Frame Leaf Length (cm) at Harvest Maturity Trail Location: Loc. 1 Loc. 2 Loc. 3 Loc. 4 Loc. 5 Loc. 6 Loc. 7 Loc. 8 Loc. 9 Loc. 10 Loc. 11 Loc. 12 VALLEY FRESH 18 19 22 25 25 21 20 17 17 20 17 18 19 17 22 22 25 25 19 20 21 20 19 19 20 19 24 19 23 24 17 19 20 18 17 21 20 16 22 22 25 27 19 19 19 18 118 19 18 19 24 23 24 28 19 20 18 19 21 18 20 18 23 21 22 24 19 19 21 19 119 20 19 19 21 22 25 24 20 19 21 17 21 19 19 18 23 19 30 27 20 20 21 17 22 17 18 18 24 22 24 25 19 19 20 20 22 20 18 19 21 23 25 28 20 18 19 20 20 19 Green Thunder 19 17 21 25 25 28 20 18 17 18 20 21 19 17 24 21 19 21 18 20 18 17 20 19 18 17 21 21 25 25 18 18 17 17 22 20 17 17 24 23 24 25 21 20 18 17 18 20 16 19 21 20 25 24 19 19 17 18 21 19 16 16 23 23 26 24 20 20 18 17 18 21 17 17 22 19 21 24 19 19 18 17 18 17 18 16 22 22 20 25 19 20 17 18 18 20 18 17 20 25 25 17 20 19 17 17 21 19 18 18 23 20 23 28 19 18 18 18 22 20 PIC Cos 19 17 22 20 22 21 19 18 20 17 21 25 20 19 23 26 22 23 21 20 17 17 21 21 18 18 21 20 24 20 17 18 16 18 21 20 18 19 23 21 20 22 21 19 18 16 19 19 20 18 20 21 21 23 17 19 17 18 20 20 18 17 21 22 22 25 20 21 19 19 19 21 19 22 18 20 19 23 19 19 17 18 21 20 20 20 21 18 24 26 21 19 19 19 22 19 19 18 20 20 22 23 18 20 17 16 23 20 18 20 21 21 24 24 20 19 20 20 22 19 Anova: Two-Factor With Replication SUMMARY Loc. 1 Loc. 2 Loc. 3 Loc. 4 Loc. 5 Loc. 6 Loc. 7 Loc. 8 Loc. 9 Loc. 10 Loc. 11 Loc. 12 VALLEY FRESH Count 10 10 10 10 10 10 10 10 10 10 10 10 Sum 189 182 226 218 248 253 192 190 197 188 396 190 Average 18.9 18.2 22.6 21.8 24.8 25.3 19.2 19 19.7 18.8 39.6 19 Variance 0.766667 1.066667 1.3777778 3.28889 4.4 4.9 0.84444 0.88889 2.0111 1.51111 1732.489 1.33333 Green Thunder Count 10 10 10 10 10 10 10 10 10 10 10 10 Sum 176 171 221 219 233 241 193 191 175 174 198 196 Average 17.6 17.1 22.1 21.9 23.3 24.1 19.3 19.1 17.5 17.4 19.8 19.6 Variance 1.155556 0.766667 1.8777778 4.32222 6.01111 10.32222 0.9 0.76667 0.2778 0.26667 2.844444 1.37778 PIC Cos Count 10 10 10 10 10 10 10 10 10 10 10 10 Sum 189 188 210 209 220 230 193 192 180 178 209 204 Average 18.9 18.8 21 20.9 22 23 19.3 19.2 18 17.8 20.9 20.4 Variance 0.766667 2.4 2.2222222 4.32222 2.88889 3.111111 2.45556 0.84444 2 1.73333 1.655556 3.15556 TOTALS VALLEY FRESH Green Thunder PIC COS Count 120 120 120 Sum 2669 2388 2402 Average 22.24167 19.9 20.01667 Variance 165.7982 7.737815 4.470308 ANOVA Source of Variation SS df MS F P-value F crit Variety 417.9056 2 208.95278 4.14835 0.016637 3.023601 Locations 2645.831 11 240.53005 4.77526 8.2E−07 1.818261 Interaction 2217.028 22 100.77399 2.00067 0.005398 1.575174 Within 16319.9 324 50.370062 Total 21600.66 359

Table 11 below shows the leaf index of Valley Fresh at harvest maturity as compared to Green Thunder and PIC Cos. Leaf index is calculated by dividing the leaf length by the leaf width. An analysis of variance was performed on the data and is shown below the data. As can be seen in Table 11, Valley Fresh has a significantly different leaf index than Green Thunder and PIC Cos at harvest maturity.

TABLE 11 Leaf index at Harvest Maturity Trail Location: Loc. 1 Loc. 2 Loc. 3 Loc. 4 Loc. 5 Loc. 6 Loc. 7 Loc. 8 Loc. 9 Loc. 10 Loc. 11 Loc. 12 VALLEY FRESH 1.78 1.74 1.36 1.16 1.52 1.62 1.80 1.94 2.06 1.85 1.82 1.78 1.58 1.88 1.23 1.36 1.40 1.28 1.79 1.80 1.62 1.75 1.84 1.84 1.60 1.63 1.33 1.47 1.48 1.54 2.00 1.79 1.85 1.89 2.00 1.76 1.45 1.75 1.23 1.36 1.32 1.33 1.74 1.79 1.89 2.00 0.29 1.68 1.67 1.63 1.17 1.22 1.58 1.29 1.89 1.75 1.89 1.89 1.67 1.89 1.65 1.61 1.35 1.48 1.45 1.46 1.84 1.89 1.71 1.79 0.29 1.80 1.74 1.63 1.43 1.23 1.44 1.33 1.70 1.84 1.62 2.00 1.76 1.79 1.63 1.72 1.22 1.58 1.20 1.37 1.80 1.70 1.67 2.06 1.55 1.88 1.61 1.83 1.13 1.36 1.42 1.44 1.74 1.79 1.85 1.80 1.55 1.90 1.67 1.63 1.38 1.30 1.36 1.36 1.70 1.83 1.84 1.80 1.75 1.79 Green Thunder 1.47 1.47 1.43 1.20 1.32 1.18 1.60 1.83 2.06 1.94 1.50 1.57 1.37 1.53 1.38 1.43 1.68 1.52 1.83 1.65 1.94 2.06 1.65 1.68 1.44 1.53 1.52 1.43 1.24 1.44 1.72 1.72 2.00 2.12 1.45 1.60 1.59 1.41 1.25 1.39 1.33 1.32 1.57 1.65 2.00 2.12 1.72 1.55 1.63 1.47 1.48 1.50 1.40 1.46 1.68 1.74 2.00 2.00 1.52 1.68 1.63 1.69 1.52 1.39 1.27 1.50 1.50 1.60 1.89 2.12 1.78 1.43 1.59 1.71 1.50 1.74 1.62 1.33 1.63 1.79 1.94 2.00 1.78 1.82 1.39 1.81 1.50 1.50 1.50 1.32 1.74 1.65 2.12 1.89 1.72 1.55 1.44 1.53 1.55 1.24 1.36 1.88 1.65 1.68 2.06 2.00 1.52 1.74 1.56 1.61 1.43 1.70 1.52 1.21 1.68 1.78 1.94 1.94 1.50 1.70 PIC Cos 1.47 1.76 1.41 1.60 1.55 1.57 1.89 2.00 1.95 2.29 1.67 1.36 1.45 1.58 1.35 1.19 1.64 1.43 1.62 1.70 2.29 2.35 1.52 1.52 1.67 1.56 1.48 1.50 1.33 1.70 2.06 1.83 2.50 2.11 1.67 1.75 1.50 1.53 1.26 1.57 1.50 1.45 1.57 1.84 2.22 2.50 1.84 1.79 1.50 1.56 1.50 1.57 1.71 1.52 2.12 1.79 2.41 2.17 1.55 1.70 1.67 1.65 1.33 1.41 1.55 1.24 1.65 1.62 2.00 2.11 1.74 1.67 1.53 1.59 1.61 1.50 1.63 1.39 1.84 1.84 2.35 2.17 1.67 1.80 1.50 1.45 1.48 1.67 1.38 1.38 1.67 1.89 2.11 2.05 1.73 1.84 1.78 1.74 1.36 1.16 1.52 1.62 1.80 1.94 2.06 1.85 1.82 1.78 1.58 1.88 1.23 1.36 1.40 1.28 1.79 1.80 1.62 1.75 1.84 1.84 Anova: Two-Factor With Replication Leaf Index (Calculated by dividing the leaf length by the leaf width) SUMMARY Loc. 1 Loc. 2 Loc. 3 Loc. 4 Loc. 5 Loc. 6 Loc. 7 Loc. 8 Loc. 9 Loc. 10 Loc. 11 Loc. 12 VALLEY FRESH Count 10 10 10 10 10 10 10 10 10 10 10 10.00 Sum 16.36959 17.06218 12.817923 13.5287 14.17281 14.01894 18 18.1298 18.004 18.8319 14.50897 18.12 Average 1.636959 1.706218 1.2817923 1.35287 1.417281 1.401894 1.8 1.81298 1.8004 1.88319 1.450897 1.81 Variance 0.008014 0.009069 0.0103358 0.01722 0.011556 0.012454 0.00876 0.00477 0.0201 0.01109 0.394856 0.00 Green Thunder Count 15.10191 15.76127 14.560093 14.5166 14.24756 14.17069 16.6138 17.0939 19.958 20.1895 16.15216 16.33 Sum 1.510191 1.576127 1.4560093 1.45166 1.424756 1.417069 1.66138 1.70939 1.9958 2.01895 1.615216 1.63 Average 0.009488 0.015726 0.0080261 0.02935 0.02251 0.040094 0.00877 0.00562 0.0047 0.00666 0.016226 0.01 Variance 15.10191 15.76127 14.560093 14.5166 14.24756 14.17069 16.6138 17.0939 19.958 20.1895 16.15216 16.33 PIC Cos Count 10 10 10 10 10 10 10 10 10 10 10 10.00 Sum 15.46754 15.7246 14.393183 14.8395 15.15647 14.59334 18.0705 17.9076 22.198 22.1244 16.49465 17.07 Average 1.546754 1.57246 1.4393183 1.48395 1.515647 1.459334 1.80705 1.79076 2.2198 2.21244 1.649465 1.71 Variance 0.007946 0.007349 0.0111824 0.01816 0.015838 0.015056 0.04066 0.01357 0.0425 0.02579 0.011872 0.02 TOTALS VALLEY FRESH Green Thunder PIC COS Count 120 120 120 Sum 193.5606 194.6938 204.044 Average 1.613005 1.622448 1.700367 Variance 0.082335 0.052172 0.085053 ANOVA Source of Variation SS df MS F P-value F crit Variety 2 0.2758501 10.7113 3.13E−05 3.023601 0.5517 Locations 11 1.4335248 55.6642 5.91E−68 1.818261 15.76877 Interaction 22 0.0915877 3.55638 2.97E−07 1.575174 2.014929 Within 324 0.0257531 8.344002 Total 26.6794 359

Table 12 below shows the leaf area of Valley Fresh as compared to Green Thunder and PIC Cos. Leaf area is calculated by multiplying the leaf length by the leaf width. An analysis of variance was performed on the data and is shown below the data. As can be seen in Table 12, Valley Fresh has a significantly larger leaf area than Green Thunder and PIC Cos.

TABLE 12 Leaf area at Harvest Maturity Trail Location: Loc. 1 Loc. 2 Loc. 3 Loc. 4 Loc. 5 Loc. 6 Loc. 7 Loc. 8 Loc. 9 Loc. 10 Loc. 11 Loc. 12 Valley Fresh 576 627 660 725 950 714 720 561 595 740 527 576 570 544 594 660 875 800 646 720 714 700 665 665 640 589 768 532 782 888 578 646 740 612 578 777 580 448 594 660 825 972 627 646 684 648 4012 608 540 589 672 644 912 1008 684 700 612 684 735 612 660 522 713 651 704 840 665 684 756 646 4046 720 627 589 630 594 900 768 680 665 714 578 777 646 589 558 644 570 1080 999 720 680 735 595 748 544 522 594 648 660 816 900 627 646 740 720 748 760 540 589 609 690 850 1064 680 594 665 720 700 646 Green Thunder 532 425 630 750 825 924 640 594 595 630 600 693 494 442 792 630 608 672 594 660 630 595 660 608 468 442 672 630 775 900 558 558 578 612 704 640 459 408 720 736 768 825 693 660 648 612 558 620 416 532 651 600 875 840 608 627 578 648 672 608 416 432 805 736 858 864 600 640 612 612 576 630 459 493 726 627 714 768 589 646 630 578 576 527 450 464 726 726 600 825 627 660 612 612 558 620 468 442 620 775 850 544 660 608 595 578 672 627 504 522 759 680 805 952 608 576 630 630 726 680 PIC Cos 532 510 682 640 748 693 684 648 780 663 735 850 580 570 713 806 792 759 714 680 663 680 672 672 540 504 651 600 768 680 595 594 640 684 735 700 486 551 667 693 600 704 693 665 720 640 665 646 600 504 600 693 756 805 612 646 697 702 620 680 540 476 588 682 748 775 660 714 722 760 627 735 551 770 522 600 589 736 665 665 680 702 735 720 600 580 651 540 792 936 735 684 760 741 836 665 608 504 620 560 726 782 648 660 697 608 782 720 486 600 630 630 792 816 660 627 780 800 792 665 Anova: Two-Factor With Replication Leaf Area SUMMARY Loc. 1 Loc. 2 Loc. 3 Loc. 4 Loc. 5 Loc. 6 Loc. 7 Loc. 8 Loc. 9 Loc. 10 Loc. 11 Loc. 12 VALLEY FRESH Count 10 10 10 10 10 10 10 10 10 10 10 10 Sum 5844 5649 6532 6386 8694 8953 6627 6542 6955 6643 13536 6554 Average 584.4 564.9 653.2 638.6 869.4 895.3 662.7 654.2 695.5 664.3 1353.6 655.4 Variance 2086.267 2557.433 2974.1778 3302.49 10425.16 13232.01 1958.46 2289.96 3111.2 3242.68 1994548 5890.49 Green Thunder Count 10 10 10 10 10 10 10 10 10 10 10 10 Sum 4666 4602 7101 6890 7678 8114 6177 6229 6108 6107 6302 6253 Average 466.6 460.2 710.1 689 767.8 811.4 617.7 622.9 610.8 610.7 630.2 625.3 Variance 1326.933 1757.511 4242.9889 3968 9742.178 15230.04 1499.34 1382.32 571.51 503.122 4024.4 2014.9 PIC Cos Count 10 10 10 10 10 10 10 10 10 10 10 10 Sum 5523 5569 6324 6444 7311 7686 6666 6583 7139 6980 7199 7053 Average 552.3 556.9 632.4 644.4 731.1 768.6 666.6 658.3 713.9 698 719.9 705.3 Variance 1992.011 7238.767 2912.7111 6049.38 5676.1 5634.267 1829.82 1077.57 2304.3 3259.78 5237.433 3421.57 TOTALS VALLEY FRESH Green Thunder PIC COS Count 120 120 120 Sum 88915 76227 80477 Average 740.9583 635.225 670.6417 Variance 198260.9 13411.57 7624.4 ANOVA Source of Variation SS df MS F P-value F crit Variety 695132.5 2 347566.23 5.85097 0.00319 3.023601 Locations 3905501 11 355045.54 5.97688 6.94E−09 1.818261 Interaction 2944195 22 133827.03 2.25286 0.001256 1.575174 Within 19246636 324 59403.197 Total 26791464 359

Table 13 below shows the core length of Valley Fresh as compared to Green Thunder and PIC Cos at harvest maturity. Core length is measured in centimeters. An analysis of variance was performed on the data and is shown below the data. As can be seen in Table 13, Valley Fresh has a significantly longer core length than Green Thunder and PIC Cos at harvest maturity.

TABLE 13 Core length (cm) Trail Location: Loc. 1 Loc. 2 Loc. 3 Loc. 4 Loc. 5 Loc. 6 Loc. 7 Loc. 8 Loc. 9 Loc. 10 Loc. 11 Loc. 12 Valley Fresh 7.0 8.0 8.0 7.0 9.5 6.2 6.2 5.6 5.0 5.8 7.0 4.6 9.5 7.0 9.0 8.0 7.0 6.0 5.5 6.3 5.5 5.2 6.7 7.5 8.0 7.5 9.0 10.0 7.9 8.5 6.5 6.2 5.8 5.2 6.9 6.5 6.0 8.5 9.0 9.5 8.2 8.0 6.2 6.5 5.5 6.0 7.0 7.0 7.5 7.5 8.0 10.0 6.5 8.2 7.0 7.2 5.5 5.5 7.1 6.2 7.5 9.0 8.5 6.5 8.0 6.6 6.9 7.2 6.0 5.5 7.6 8.1 7.5 7.5 8.5 8.0 7.7 7.1 6.1 6.4 5.0 5.3 7.1 7.2 9.0 8.5 8.0 9.0 7.6 8.7 6.6 6.2 5.5 5.5 6.5 6.5 7.5 9.5 11.0 9.0 10.2 7.5 5.8 6.5 5.5 5.8 6.0 7.6 7.5 7.0 6.0 7.0 8.7 8.9 6.0 5.9 5.3 5.8 8.5 7.1 Green Thunder 8.0 5.5 8.0 8.5 5.7 8.7 5.5 5.0 6.0 5.9 6.7 6.5 8.0 5.0 7.5 8.5 4.7 5.7 6.0 5.9 6.2 5.4 7.5 7.2 7.5 6.0 7.0 7.5 8.6 8.4 4.8 6.0 5.0 6.0 7.0 8.0 8.0 5.0 7.5 5.0 7.2 6.4 6.0 5.6 5.9 5.7 6.0 8.0 8.5 7.0 7.0 7.5 8.5 6.5 5.3 6.0 4.6 5.1 7.0 6.2 6.5 6.0 7.0 7.5 6.4 6.4 5.5 5.3 4.8 6.1 7.0 7.7 5.0 5.5 8.0 9.5 7.5 6.6 5.9 6.0 5.3 6.0 8.5 7.5 7.5 5.0 6.0 6.0 5.6 8.5 6.0 5.5 6.1 4.6 6.5 8.0 6.5 7.0 5.0 8.0 5.6 7.0 5.6 5.8 6.0 5.0 9.0 6.6 7.5 6.5 6.0 7.5 7.0 5.8 6.0 5.8 5.8 5.9 6.0 6.9 PIC Cos 5.0 6.0 7.0 7.0 6.6 5.6 6.5 6.8 4.8 4.6 7.0 6.2 6.5 8.0 8.0 6.5 6.5 6.6 6.0 6.6 5.5 5.1 6.4 8.1 7.0 6.5 8.0 8.5 5.5 3.6 5.8 5.9 4.7 4.6 6.1 6.2 7.5 9.0 7.5 8.0 6.0 4.7 6.7 6.5 4.5 4.8 6.6 6.9 8.5 7.5 8.0 7.5 6.5 5.6 6.8 6.0 5.0 4.8 7.2 6.1 6.0 9.0 7.0 8.0 4.5 6.4 6.5 6.7 5.3 4.7 7.1 7.0 8.0 8.0 5.5 8.0 6.0 5.6 6.3 6.2 4.9 4.8 6.2 7.2 9.0 8.0 8.0 8.0 4.5 7.5 6.6 6.0 5.2 4.7 6.4 6.8 6.5 8.0 7.0 7.5 6.5 6.4 6.5 6.6 4.5 5.4 5.9 6.3 7.0 8.0 9.0 6.5 5.1 4.7 6.0 6.5 4.6 4.7 6.1 6.0 Anova: Two-Factor With Replication Core length (cm) SUMMARY Loc. 1 Loc. 2 Loc. 3 Loc. 4 Loc. 5 Loc. 6 Loc. 7 Loc. 8 Loc. 9 Loc. 10 Loc. 11 Loc. 12 VALLEY FRESH Count 10 10 10 10 10 10 10 10 10 10 10 10.0 Sum 77 80 85 84 81.3 75.7 62.8 64 54.6 55.6 70.4 68.3 Average 7.7 8 8.5 8.4 8.13 7.57 6.28 6.4 5.46 5.56 7.04 6.8 Variance 0.955556 0.722222 1.5555556 1.65556 1.217889 1.111222 0.224 0.25333 0.096 0.07822 0.440444 0.9 Green Thunder Count 10 10 10 10 10 10 10 10 10 10 10 10.0 Sum 73 58.5 69 75.5 66.8 70 56.6 56.9 55.7 55.7 71.2 72.6 Average 7.3 5.85 6.9 7.55 6.68 7 5.66 5.69 5.57 5.57 7.12 7.3 Variance 1.066667 0.613889 0.9333333 1.63611 1.704 1.262222 0.16044 0.11433 0.349 0.26678 0.966222 0.5 PIC Cos Count 10 10 10 10 10 10 10 10 10 10 10 10.0 Sum 71 78 75 75.5 57.7 56.7 63.7 63.8 49 48.2 65 66.8 Average 7.1 7.8 7.5 7.55 5.77 5.67 6.37 6.38 4.9 4.82 6.5 6.7 Variance 1.433333 0.9 0.8888889 0.46944 0.682333 1.273444 0.11122 0.10622 0.12 0.06178 0.211111 0.4 TOTALS VALLEY FRESH Green Thunder PIC COS Count 120 120 120 Sum 858.7 781.5 770.4 Average 7.155833 6.5125 6.42 Variance 1.738789 1.280767 1.408504 ANOVA Source of Variation SS df MS F P-value F crit Variety 38.55539 2 19.277694 27.2431 1.16E−11 3.023601 Locations 224.2859 11 20.389626 28.8145 7.85E−42 1.818261 Interaction 73.38528 22 3.3356944 4.71398 1.2E−10 1.575174 Within 229.268 324 0.7076173 Total 565.4946 359

Table 14 below shows the core diameter of Valley Fresh as compared to Green Thunder and PIC Cos at harvest maturity. Core diameter is measured in centimeters. An analysis of variance was performed on the data and is shown below the data. As can be seen in Table 14, Valley Fresh has a significantly different core diameter than Green Thunder and PIC Cos.

TABLE 14 Core diameter (cm) Trail Location: Loc. 1 Loc. 2 Loc. 3 Loc. 4 Loc. 5 Loc. 6 Loc. 7 Loc. 8 Loc. 9 Loc. 10 Loc. 11 Loc. 12 Valley Fresh 4.0 4.0 4.0 4.5 4.0 4.5 3.0 3.4 3.5 3.5 3.4 3.1 4.0 4.0 4.5 5.0 3.2 2.5 3.5 3.4 3.4 3.6 3.6 3.4 4.0 4.0 5.0 4.5 3.5 4.7 3.2 3.0 3.6 3.4 3.4 3.6 4.0 4.0 4.5 4.5 4.4 4.2 4.0 4.0 3.7 3.5 3.3 3.2 4.0 3.5 4.5 5.0 4.4 3.7 3.5 3.3 3.5 3.6 3.5 3.8 4.0 4.0 5.0 3.5 4.2 4.0 3.5 3.6 3.5 3.6 3.9 4.0 4.5 4.0 4.5 4.0 4.0 4.0 3.0 3.4 3.5 3.6 3.2 3.6 4.0 4.0 4.5 4.0 3.5 4.0 3.4 4.0 3.5 3.2 3.5 3.6 4.5 4.0 4.5 3.5 4.5 4.0 3.7 3.3 3.6 3.7 3.8 3.8 4.0 3.5 5.0 4.0 4.0 4.5 4.0 3.6 3.4 3.6 4.1 3.3 Green Thunder 4.0 3.5 4.5 4.0 4.2 4.6 3.5 3.3 3.7 3.6 3.7 3.5 4.0 4.0 4.5 4.0 3.5 3.4 3.8 3.7 3.5 3.5 3.8 3.8 4.0 4.0 3.5 3.5 4.2 4.0 3.2 3.8 3.5 3.7 3.8 3.8 3.0 4.0 4.0 3.5 4.5 4.5 3.5 3.6 3.8 3.8 3.5 3.5 3.5 4.0 4.0 3.5 4.0 4.5 3.8 3.8 3.6 3.8 3.5 3.6 3.5 3.5 3.5 3.5 4.0 4.0 3.5 3.8 3.5 3.7 3.5 3.8 3.5 4.0 4.0 4.0 4.0 3.8 3.2 3.6 3.8 3.6 3.8 3.8 4.0 3.5 3.5 4.0 4.1 4.0 3.8 3.5 3.5 3.6 3.4 3.9 3.5 4.0 3.5 4.0 3.3 4.2 3.6 3.7 3.8 3.5 3.5 3.7 3.5 4.0 3.5 3.5 3.9 3.6 3.6 3.7 3.7 3.5 3.2 3.7 PIC Cos 4.0 4.5 4.0 4.0 4.2 4.0 4.0 4.0 3.5 4.0 4.0 3.9 4.0 5.0 4.0 3.5 3.7 3.4 4.2 4.8 4.0 3.9 3.5 4.1 5.0 5.0 4.0 4.0 4.0 2.7 4.0 3.8 3.8 4.0 4.1 3.6 5.0 5.0 4.0 4.0 4.2 3.0 4.8 4.0 4.0 3.9 4.1 3.1 5.0 5.0 4.0 4.0 4.2 4.0 4.0 4.2 4.0 3.7 3.6 4.0 5.5 5.0 4.0 4.5 3.5 3.2 4.2 4.7 3.9 4.0 3.8 3.9 5.0 5.0 4.0 4.0 3.6 3.7 3.8 4.2 3.5 3.8 4.0 3.8 5.5 5.0 4.0 4.5 4.7 3.3 4.4 4.0 4.0 3.6 3.6 4.0 5.0 5.0 4.0 4.0 4.0 3.6 4.2 4.8 3.7 4.0 3.9 3.9 5.0 4.5 4.0 3.5 4.0 3.0 4.2 4.2 3.7 3.8 3.9 3.9 Anova: Two-Factor With Replication Core diameter (cm) SUMMARY Loc. 1 Loc. 2 Loc. 3 Loc. 4 Loc. 5 Loc. 6 Loc. 7 Loc. 8 Loc. 9 Loc. 10 Loc. 11 Loc. 12 VALLEY FRESH Count 10 10 10 10 10 10 10 10 10 10 10 10 Sum 41 39 46 42.5 39.7 40.1 34.8 35 35.2 35.3 35.7 35.4 Average 4.1 3.9 4.6 4.25 3.97 4.01 3.48 3.5 3.52 3.53 3.57 3.54 Variance 0.044444 0.044444 0.1 0.29167 0.193444 0.374333 0.12622 0.09778 0.0084 0.02011 0.080111 0.08267 Green Thunder Count 10 10 10 10 10 10 10 10 10 10 10 10 Sum 36.5 38.5 38.5 37.5 39.7 40.6 35.5 36.5 36.4 36.3 35.7 37.1 Average 3.65 3.85 3.85 3.75 3.97 4.06 3.55 3.65 3.64 3.63 3.57 3.71 Variance 0.113889 0.058333 0.1694444 0.06944 0.120111 0.158222 0.04944 0.025 0.0182 0.01344 0.040111 0.01878 PIC Cos Count 10 10 10 10 10 10 10 10 10 10 10 10 Sum 49 49 40 40 40.1 33.9 41.8 42.7 38.1 38.7 38.5 38.2 Average 4.9 4.9 4 4 4.01 3.39 4.18 4.27 3.81 3.87 3.85 3.82 Variance 0.266667 0.044444 0 0.11111 0.123222 0.189889 0.07511 0.13344 0.041 0.02011 0.047222 0.08178 TOTALS VALLEY FRESH Green Thunder PIC COS Count 120 120 120 Sum 459.7 448.8 490 Average 3.830833 3.74 4.083333 Variance 0.234083 0.088471 0.26409 ANOVA Source of Variation SS df MS F P-value F crit Variety 7.595389 2 3.7976944 39.6039 4.1E−16 3.023601 Locations 15.84564 11 1.4405126 15.0222 1.46E−23 1.818261 Interaction 22.89594 22 1.0407247 10.8531 1.41E−27 1.575174 Within 31.069 324 0.095892 Total 77.40597 359

Table 15 below shows the seed stalk height for Valley Fresh as compared to the seed stalk height for Green Thunder and PIC Cos at harvest maturity. Seed stalk height is measured in centimeters. An analysis of variance was performed on the data and is shown below the data. As can be seen from the data in Table 15, Valley Fresh has a significantly taller seed stalk height than PIC Cos.

TABLE 15 Seed Stalk Height (cm) Valley Fresh Green Thunder PIC Cos 100 105 100 102 107 102 105 109 100 100 108  95 110 100  96  95 102 105 100 103  97 110 111  98 108 110  99 105 101  98 102 102  99 102 100 100 104 110  96 Anova: Single Factor SUMMARY Groups Count Sum Average Variance VALLEY FRESH 13 1343 103.3077 18.73077 Green Thunder 13 1368 105.2308 16.85897 PIC Cos 13 1285 98.84615 7.307692 ANOVA Source of Variation SS df MS F P-value F crit Between Varieties 278.9231 2 139.4615 9.753138 0.000412 3.259444 Within Varieties 514.7692 36 14.29915 Total 793.6923 38

DEPOSIT INFORMATION

A deposit of the Synergene Seed & Technology, Inc. proprietary lettuce cultivar designated Valley Fresh disclosed above and recited in the appended claims has been made with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110. The date of deposit was Aug. 27, 2008. The deposit of 2,500 seeds was taken from the same deposit maintained by Synergene Seed & Technology, Inc. since prior to the filing date of this application. All restrictions upon the deposit have been removed, and the deposit is intended to meet all of the requirements of 37 C.F.R. 1.801-1.809. The ATCC accession number is PTA-9438. The deposit will be maintained in the depository for a period of 30 years, or 5 years after the last request, or for the effective life of the patent, whichever is longer, and will be replaced as necessary during that period.

While a number of exemplary aspects and embodiments have been discussed above, those of skill in the art will recognize certain modifications, permutations, additions and sub-combinations thereof. It is therefore intended that the following appended claims and claims hereafter introduced are interpreted to include all such modifications, permutations, additions and sub-combinations as are within their true spirit and scope. 

1. A seed of lettuce cultivar Valley Fresh, representative sample seed of said cultivar was deposited under ATCC Accession No. PTA-9438.
 2. A lettuce plant, or a part thereof, produced by growing the seed of claim
 1. 3. A tissue culture produced from protoplasts or cells from the plant of claim 2, wherein said cells or protoplasts of the tissue culture are produced from a plant part selected from the group consisting of leaf, pollen, embryo, cotyledon, hypocotyl, meristematic cell, root, root tip, pistil, anther, flower, shoot, stem, seed, and petiole.
 4. A lettuce plant regenerated from the tissue culture of claim 3, wherein the plant has all of the morphological and physiological characteristics of cultivar Valley Fresh.
 5. A method for producing a lettuce seed comprising crossing two lettuce plants and harvesting the resultant lettuce seed, wherein at least one lettuce plant is the lettuce plant of claim
 2. 6. A lettuce seed produced by the method of claim
 5. 7. A lettuce plant, or a part thereof, produced by growing said seed of claim
 6. 8. A method of producing a male sterile lettuce plant wherein the method comprises transforming the lettuce plant of claim 2 with a nucleic acid molecule that confers male sterility.
 9. A male sterile lettuce plant produced by the method of claim
 8. 10. A method of producing an herbicide resistant lettuce plant wherein the method comprises transforming the lettuce plant of claim 2 with a transgene wherein the transgene confers resistance to an herbicide selected from the group consisting of imidazolinone, sulfonylurea, glyphosate, glufosinate, L-phosphinothricin, triazine and benzonitrile.
 11. An herbicide resistant lettuce plant produced by the method of claim
 10. 12. A method of producing a pest or insect resistant lettuce plant wherein the method comprises transforming the lettuce plant of claim 2 with a transgene that confers pest or insect resistance.
 13. A pest or insect resistant lettuce plant produced by the method of claim
 12. 14. The lettuce plant of claim 13, wherein the transgene encodes a Bacillus thuringiensis endotoxin.
 15. A method of producing a disease resistant lettuce plant wherein the method comprises transforming the lettuce plant of claim 2 with a transgene that confers disease resistance.
 16. A disease resistant lettuce plant produced by the method of claim
 15. 17. A method of producing a lettuce plant with a value-added trait, wherein the method comprises transforming the lettuce plant of claim 2 with a transgene encoding a protein selected from the group consisting of a ferritin, a nitrate reductase, and a monellin.
 18. A lettuce plant with a value-added trait produced by the method of claim
 17. 19. A method of introducing a desired trait into lettuce cultivar Valley Fresh wherein the method comprises: (a) crossing a Valley Fresh plant, wherein a representative sample of seed was deposited under ATCC Accession No. PTA-9438, with a plant of another lettuce cultivar that comprises a desired trait to produce progeny plants wherein the desired trait is selected from the group consisting of male sterility, herbicide resistance, insect or pest resistance, modified bolting and resistance to bacterial disease, fungal disease or viral disease; (b) selecting one or more progeny plants that have the desired trait to produce selected progeny plants; (c) crossing the selected progeny plants with the Valley Fresh plants to produce backcross progeny plants; (d) selecting for backcross progeny plants that have the desired trait and all of the physiological and morphological characteristics of lettuce cultivar Valley Fresh listed in Table 1; and (e) repeating steps (c) and (d) three or more times in succession to produce selected fourth or higher backcross progeny plants that comprise the desired trait and all of the physiological and morphological characteristics of lettuce cultivar Valley Fresh listed in Table
 1. 20. A lettuce plant produced by the method of claim 19, wherein the plant has the desired trait.
 21. The lettuce plant of claim 20, wherein the desired trait is herbicide resistance and the resistance is conferred to an herbicide selected from the group consisting of imidazolinone, sulfonylurea, glyphosate, glufosinate, L-phosphinothricin, triazine and benzonitrile.
 22. The lettuce plant of claim 20, wherein the desired trait is insect or pest resistance and the insect or pest resistance is conferred by a transgene encoding a Bacillus thuringiensis endotoxin. 